Manufacture of antisense oligomers and conjugates targeting PCSK9

ABSTRACT

The present invention relates to oligomeric compounds and conjugates thereof that target Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) PCSK9 mRNA in a cell, leading to reduced expression of PCSK9. Reduction of PCSK9 expression is beneficial for a range of medical disorders, such as hypercholesterolemia and related disorders.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/836,144, filed Dec. 8, 2017, which is a continuation of U.S.application Ser. No. 14/897,223, filed Dec. 9, 2015, which was theNational Stage under 35 C.F.R. § 371 of International Application No.PCT/EP2014/063757, filed Jun. 27, 2014, which claims the benefit of EPApplication No. 13174092.0, filed Jun. 27, 2013, EP Application No.13192930.9, filed Nov. 14, 2013, EP Application No. 13192938.2, filedNov. 14, 2013, EP Application No. 14153253.1, filed Jan. 30, 2014, EPApplication No. 14168331.8, filed May 14, 2014, and InternationalApplication No. PCT/EP2013/073858, filed Nov. 14, 2013, all of which areincorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:4009.0030008_Sequence_listing_ST25.txt; Size: 26,479 bytes; and Date ofCreation: May 4, 2018) filed with the application is incorporated hereinby reference in its entirety.

FIELD OF INVENTION

The present invention relates to oligomeric compounds and conjugatesthereof that target Proprotein Convertase Subtilisin/Kexin type 9(PCSK9) mRNA in a cell, leading to reduced expression of PCSK9.Reduction of PCSK9 expression is beneficial for a range of medicaldisorders, such as hypercholesterolemia and related disorders.

BACKGROUND

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as atherapeutic target for the reduction of low-density lipoproteincholesterol (LDL-C). PCSK9 increases the degradation of the LDLreceptor, resulting in high LDL-C in individuals with high PCSK9activity.

Lindholm et al., Molecular Therapy (2012); 20 2, 376-381 reports on twoLNA antisense oligonucleotides targeting PCSK9 that produce sustainedreduction of LDL-C in nonhuman primates after a loading dose (20 mg/kg)and four weekly maintenance doses (5 mg/kg). The compounds used were a14mer SPC5001 (SEQ ID NO 1) and a 13mer SPC4061. SPC5001 is likewisedisclosed in WO2011/009697. The efficacy of these PCSK9 inhibitors hasbeen attributed to their short length (Krieg et al., Molecular TherapyNucleic Acids (2012) 1, e6).

WO2007/146511 reports on short bicyclic (LNA) gapmer antisenseoligonucleotides which apparently are more potent and less toxic thanlonger compounds. The exemplified compounds appear to be 14 nts inlength.

According to van Poelgeest et al., (American Journal of Kidney Disease,2013 October; 62(4):796-800), the administration of LNA antisenseoligonucleotide SPC5001 in human clinical trials may result in acutekidney injury.

According to EP 1 984 381B1, Seth et al., Nucleic Acids Symposium Series2008 No. 52 553-554 and Swayze et al., Nucleic Acid Research 2007, vol35, pp 687-700, LNA oligonucleotides cause significant hepatotoxicity inanimals. According to WO2007/146511, the toxicity of LNAoligonucleotides may be avoided by using LNA gapmers as short as 12-14nucleotides in length. EP 1 984 381B1 recommends using 6′ substitutedbicyclic nucleotides to decrease the hepatotoxicity potential of LNAoligonucleotides. According to Hagedorn et al., Nucleic AcidTherapeutics 2013, the hepatotoxic potential of antisenseoligonucleotide may be predicted from their sequence and modificationpattern.

Oligonucleotide conjugates have been extensively evaluated for use insiRNAs, where they are considered essential in order to obtainsufficient in vivo potency. For example, see WO2004/044141 refers tomodified oligomeric compounds that modulate gene expression via an RNAinterference pathway. The oligomeric compounds include one or moreconjugate moieties that can modify or enhance the pharmacokinetic andpharmacodynamic properties of the attached oligomeric compound.

WO2012/083046 reports on a galactose cluster-pharmacokinetic modulatortargeting moiety for siRNAs.

In contrast, single stranded antisense oligonucleotides are typicallyadministered therapeutically without conjugation or formulation. Themain target tissues for antisense oligonucleotides are the liver and thekidney, although a wide range of other tissues are also accessible bythe antisense modality, including lymph node, spleen, and bone marrow.

WO 2005/086775 refers to targeted delivery of therapeutic agents tospecific organs using a therapeutic chemical moiety, a cleavable linkerand a labeling domain. The cleavable linker may be, for example, adisulfide group, a peptide or a restriction enzyme cleavableoligonucleotide domain.

WO 2011/126937 refers to targeted intracellular delivery ofoligonucleotides via conjugation with small molecule ligands.

WO2009/025669 refers to polymeric (polyethylene glycol) linkerscontaining pyridyl disulphide moieties. See also Zhao et al.,Bioconjugate Chem. 2005 16 758-766.

Chaltin et al., Bioconjugate Chem. 2005 16 827-836 reports oncholesterol modified mono-di- and tetrameric oligonucleotides used toincorporate antisense oligonucleotides into cationic liposomes, toproduce a dendrimeric delivery system. Cholesterol is conjugated to theoligonucleotides via a lysine linker.

Other non-cleavable cholesterol conjugates have been used to targetsiRNAs and antagomirs to the liver—see for example, Soutscheck et al.,Nature 2004 vol. 432 173-178 and Krützfeldt et al., Nature 2005 vol 438,685-689. For the partially phosphorothiolated siRNAs and antagomirs, theuse of cholesterol as a liver targeting entity was found to be essentialfor in vivo activity.

OBJECTIVE OF THE INVENTION

There is therefore a need for PCSK9 targeting antisense compounds, whichare as effective as SPC5001, but have a reduced toxicity risk, inparticular reduced kidney toxicity.

According to the present invention this has been achieved byidentification of new human PCSK9 sequences which are particularlyeffective to target using the antisense approach (SEQ ID NO 33 and SEQID NO 34), as well as longer variants of the SPC5001 sequence whichretain or are improved over the remarkable potency of SPC5001 withouttoxicity issues. The antisense oligonucleotides of the invention may befurther improved by using conjugates, which have been found to greatlyenhance the therapeutic index of LNA antisense oligonucleotides.

The compounds of the present invention are potent and non-toxicinhibitors of PCSK9, useful for in treatment of hypercholesterolemia andrelated disorders.

SUMMARY OF INVENTION

The oligomer of the invention may comprise between 10-22, such as 12-18nucleotides in length, which either comprises a) contiguous sequence of10-16 nucleotides which are complementary to a corresponding length ofSEQ ID NO 33 or 34 or 45, or b) a contiguous sequence of 16 nucleotideswhich are complementary to a corresponding length of SEQ ID NO 31.

The invention provides for an antisense oligonucleotide conjugatecomprising the oligomer according to the invention, and at least onenon-nucleotide or non-polynucleotide moiety covalently attached to saidoligomer.

The invention also provides for an antisense oligonucleotide conjugatecomprising the oligomer (A) according to the invention, and at least onenon-nucleotide or non-polynucleotide moiety covalently attached to saidoligomer (C), optionally via a linker region (B and/or Y) positionedbetween the contiguous sequence of the oligomer and the conjugatemoiety.

In some embodiments, the invention also provides for an antisenseoligonucleotide conjugate comprising an oligomer of 10-22, such as 12-18nucleotides in length, wherein said oligomer comprises a) a contiguoussequence of 10-16 nucleotides which are complementary to a correspondinglength of SEQ ID NO 33 or 34, or 45 or b) a contiguous sequence of 16nucleotides which are complementary to a corresponding length of SEQ IDNO 31.

The invention also provides for a compound selected from the groupconsisting of SEQ ID NO 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,17, 16, 18, 19, 20, 21, 22, 23, and 24.

The invention provides for a pharmaceutical composition comprising theoligomer or the conjugate according to the invention, and apharmaceutically acceptable diluent, carrier, salt or adjuvant.

The invention provides for an oligomer or conjugate or pharmaceuticalcomposition according to the invention, for use as a medicament, such asfor the treatment of hypercholesterolemia or related disorder, such as adisorder selected from the group consisting of atherosclerosis,hyperlipidemia, hypercholesterolemia, familiar hypercholesterolemia e.g.gain of function mutations in PCSK9, HDL/LDL cholesterol imbalance,dyslipidemias, e.g., familial hyperlipidemia (FCHL) or familialhypercholesterolemia (FHC), acquired hyperlipidemia, statin-resistanthypercholesterolemia, coronary artery disease (CAD), and coronary heartdisease (CHD).

The invention provides for the use of an oligomer or conjugate orpharmaceutical composition of the invention, for the manufacture of amedicament for the treatment of hypercholesterolemia or a relateddisorder, such as a disorder selected from the group consisting ofatherosclerosis, hyperlipidemia, hypercholesterolemia, familiarhypercholesterolemia e.g. gain of function mutations in PCSK9, HDL/LDLcholesterol imbalance, dyslipidemias, e.g., familial hyperlipidemia(FCHL) or familial hypercholesterolemia (FHC), acquired hyperlipidemia,statin-resistant hypercholesterolemia, coronary artery disease (CAD),and coronary heart disease (CHD).

The invention provides for a method of treating hypercholesterolemia ora related disorder, such as a disorder selected from the groupconsisting atherosclerosis, hyperlipidemia, hypercholesterolemia,familiar hypercholesterolemia e.g. gain of function mutations in PCSK9,HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familialhyperlipidemia (FCHL) or familial hypercholesterolemia (FHC), acquiredhyperlipidemia, statin-resistant hypercholesterolemia, coronary arterydisease (CAD), and coronary heart disease (CHD), said method comprisingadministering an effective amount of an oligomer or conjugate orpharmaceutical composition according to the invention, to a patientsuffering from, or likely to suffer from hypercholesterolemia or arelated disorder.

The invention provides for an in vivo or in vitro method for theinhibition of PCSK9 in a cell which is expressing PCSK9, said methodcomprising administering an oligomer or conjugate or pharmaceuticalcomposition according to the invention to said cell so as to inhibitPCSK9 in said cell.

The invention also provides for an oligomer according to the invention,such as an LNA oligomer, comprising a contiguous region of 10-22, suchas 12-18, such as 13, 14, 15, 16 or 17 phosphorothioate linkednucleosides, (i.e. region A, which typically is complementary to acorresponding region of the target sequence, such as SEQ ID NO 46) andfurther comprising between 1 and 6 DNA nucleosides which are contiguouswith the LNA oligomer, wherein the inter-nucleoside linkages between theDNA, and/or adjacent to the DNA nucleoside(s), is physiologicallylabile, such as is/are phosphodiester linkages. Such an LNA oligomer maybe in the form of a conjugate, as described herein, or may, for examplebe an intermediate to be used in a subsequent conjugation step. Whenconjugated, the conjugate may, for example be or comprise a sterol, suchas cholesterol or tocopherol, or may be or comprise a (non-nucleotide)carbohydrate, such as a GalNAc conjugate, or another conjugate asdescribed herein.

The invention also provides a gapmer oligomer which comprises at leastone cET, such as (S)-cET nucleotides, of between 10-22, such as 12-18,such as 13, 14, 15, 16 or 17 nucleotides in length, which targets (i.e.has a sequence which is complementary to a corresponding part of) humanPCSK9.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Examples of tri-GalNAc conjugates which may be used. Conjugates1-4 illustrate 4 suitable GalNAc conjugate moieties, and conjugates1a-4a refer to the same conjugates with an additional linker moiety (Y)which is used to link the conjugate to the oligomer (region A or to abio-cleavable linker, such as region B). The wavy line represents thecovalent link to the oligomer.

FIG. 2: Examples of cholesterol and tocopherol conjugate moieties.Conjugates 5a and 6a refer to the same conjugates with an additionallinker moiety (Y) which is used to link the conjugate to the oligomer(region A or to a bio-cleavable linker, such as region B). The wavy linerepresents the covalent link to the oligomer.

FIG. 3: Specific LNA compounds. Beta-D-oxy LNA are identified by asuperscript ^(L) after the letter, subscript _(s) represents aphosphorothioate linkage, superscript ^(Me) preceding a capital Crepresents 5-methyl cytosine LNA, non LNA nucleotides are DNAnucleotides (no superscript L).

FIG. 4: Examples of cholesterol conjugates of the LNA compounds.Beta-D-oxy LNA are identified by a superscript ^(L) after the letter,subscript _(s) represents a phosphorothioate linkage, _(o) subscriptrepresents a phosphodiester linkage, superscript ^(Me) preceding acapital C represents 5-methyl cytosine LNA, non LNA nucleotides are DNAnucleotides (no superscript L).

FIG. 5: Examples of GalNAc conjugates of the LNA compounds. Theconjugates essentially correspond to Conj2a in Figure where the wavyline is substituted with the LNA oligomer. Beta-D-oxy LNA are identifiedby a superscript ^(L) after the letter, subscript _(s) represents aphosphorothioate linkage, superscript ^(Me) preceding a capital Crepresents 5-methyl cytosine LNA, non LNA nucleotides are DNAnucleotides (no superscript L).

FIG. 5A: Detailed structure of SEQ ID NO 18

FIG. 5B: Detailed structure of SEQ ID NO 19

FIG. 6: Example of FAM conjugate group.

FIG. 7: LNA-FAM conjugates with and without cleavable phophodiesterlinkages. Beta-D-oxy LNA are identified by a superscript ^(L) after theletter, subscript _(s) represents a phosphorothioate linkage, _(o)subscript represents a phosphodiester linkage, superscript ^(Me)preceding a capital C represents 5-methyl cytosine LNA, non LNAnucleotides are DNA nucleotides (no superscript L).

FIG. 8: Anti-PCSK9 gapmers ranked for potency in vitro.

FIG. 9: Selected anti-PCSK9 gapmers ranked for potency in vitro.

FIG. 10: In vitro potency of selected anti-PCSK9 compounds and IC50calculations.

FIG. 11: In vivo ALT data for selected anti-PCSK9 conjugates.

FIG. 12: Non-limiting Illustration of compounds of the invention. Theinter-nucleoside linkage L may be, for example phosphodiester,phosphorothioate, phosphorodithioate, boranophosphate ormethylphosphonate, such as phosphodiester. PO is a phosphodiesterlinkage. Compound a) has a region B with a single DNA (or RNA), thelinkage between the second and the first region is PO. Compound b) hastwo DNA/RNA (such as DNA) nucleosides linked by a phosphodiesterlinkage. Compound c) has three DNA/RNA (such as DNA) nucleosides linkedby a phosphodiester linkages. In some embodiments, Region B may befurther extended by further phosphodiester DNA/RNA (such as DNAnucleosides). The conjugate group (Marked X, otherwise region C herein)is illustrated on the left side of each compound (e.g. Cholesterol,GalNAc, Conj1-4, 1a-4a, and 5 or 6), and may, optionally be covalentlyattached to the terminal nucleoside of region B via a phosphorusnucleoside linkage group, such as phosphodiester, phosphorothioate,phosphorodithioate, boranophosphate or methylphosphonate, or may belinked via an alternative linkage, e.g. a triazol linkage (see L incompounds d), e), and f

FIG. 13. Non-limiting Illustration of compounds of the invention, wherethe compounds comprise the optional linker (Y) between the third(conjugate) region (X) and the second region (region B). Samenomenclature as FIG. 12. Suitable linkers are disclosed herein, andinclude, for example alkyl linkers, for example C6 linkers. In compoundsa), b) and c), the linker between X and region B is attached to region Bvia a phosphorus nucleoside linkage group, such as phosphodiester,phosphorothioate, phosphorodithioate, boranophosphate ormethylphosphonate, or may be linked via an alternative linkage eg. atriazol linkage (Li). In these compounds Lii represents theinternucleoside linkage between the first (A) and second regions (B).Compounds d), e), & f) further comprise a linker (Y) between region Band the conjugate group, and region Y may be linked to region B via, forexample, a phosphorus nucleoside linkage group, such as phosphodiester,phosphorothioate, phosphorodithioate, boranophosphate ormethylphosphonate, or in some embodiments a triazole linkage. Inaddition, or alternatively X may be an activation group or a reactivegroup. X may be covalently attached to region B via a phosphorusnucleoside linkage group, such as phosphodiester, phosphorothioate,phosphorodithioate, boranophosphate or methylphosphonate, or may belinked via an alternative linkage, e.g. a triazol linkage.

FIG. 14. Silencing of PCSK9 mRNA with cholesterol-conjugates in vivo.Mice were injected with a single dose of 10 mg/kg unconjugatedLNA-antisense oligonucleotide (#40) or equimolar amounts of LNAantisense oligonucleotides conjugated to Cholesterol with differentlinkers and sacrificed at days 1, 3, 7 and 10 after dosing. RNA wasisolated from liver and kidney and subjected to PCSK9 specific RT-qPCRA. Quantification of PCSK9 mRNA from liver samples normalized to BACTand shown as percentage of the average of equivalent saline controls B.Quantification of PCSK9 mRNA from kidney samples normalized to BACT andshown as percentage of the average of equivalent saline controls.

FIG. 15. Kim-1 expression from rat safety study (see Example 5).

FIG. 16: Serum PCSK9 and LDL cholesterol in samples from cynomolgusmonkeys injected four times (one injection/week) with 0.5 or 1.5mg/kg/week of SEQ ID 2 and 18.

FIG. 17. Serum PCSK9 and LDL cholesterol in samples from cynomolgusmonkeys injected four times (one injection/week) with 0.5 or 1.5mg/kg/week of SEQ ID 3 and 19.

DETAILED DESCRIPTION OF INVENTION

In the following different elements of the invention are described underseparate headings.

It is understood that an embodiment from one element can be combinedwith embodiments from the other elements to arrive at a compound of theinvention (e.g. as illustrated in FIGS. 12 and 13)

The Oligomer (Region A)

The term “oligomer” or “oligonucleotide” in the context of the presentinvention, refers to a molecule formed by covalent linkage of two ormore nucleotides (i.e. an oligonucleotide). Herein, a single nucleotide(unit) may also be referred to as a monomer or unit. In someembodiments, the terms “nucleoside”, “nucleotide”, “unit” and “monomer”are used interchangeably. It will be recognized that when referring to asequence of nucleotides or monomers, what is referred to is the sequenceof bases, such as A, T, G, C or U.

The oligomer of the invention may comprise between 10-22, such as 12-22nucleotides, such as 12-18 nucleotides in length. The oligomer compriseseither a) a contiguous sequence of 10-16 nucleotides which arecomplementary to a corresponding length of SEQ ID NO 33 or 34 or 45, orb) a contiguous sequence of 16 nucleotides which are complementary to acorresponding length of SEQ ID NO 31.

In some embodiments, the oligomer of the invention comprises acontiguous sequence selected from the group consisting of SEQ ID NO 26,27, 28, 29 and 44.

The compound (e.g. oligomer or conjugate) of the invention targetsPCSK9, and as such is capable of down regulating the expression of orinhibiting PCSK9, such as PCSK9 in a human or in a cell expressingPCSK9.

In some embodiments, the internucleoside linkages of the a contiguoussequence of 10-16 nucleotides which are complementary to a correspondinglength of SEQ ID NO 33 or 34 or 45 may be phosphorothioate linkages.

In some embodiments, the oligomer of the invention comprises or consistsa contiguous sequence selected from the group consisting of SEQ ID NO 2,3, 4, 5, 6, 7, 8 and 40. In one embodiment, the oligomer comprises orconsists of a sequence selected from a) SEQ ID NO 2 or 3, or b) SEQ IDNO 4, 5 or 6, or c) SEQ ID NO 7 or 8, or d) SEQ ID NO 40.

In some embodiments, the oligomer comprises 10-16 phosporothiolatelinked nucleosides.

In some embodiments, the oligomer of the invention comprises acontiguous sequence of at least 10-16 nucleotides which arecomplementary to a corresponding length of SEQ ID NO 33 or 34 or 45 or acontiguous sequence of 16 nucleotides which are complementary to acorresponding length of SEQ ID NO 31, wherein the contiguous sequencecomprises nucleotide analogues. Preferably, the nucleotide analogues areaffinity enhancing nucleotide analogues.

In some embodiments, the nucleotide analogues are sugar modifiednucleotides, such as sugar modified nucleotides independently ordependently selected from the group consisting of: Locked Nucleic Acid(LNA) units; 2′-O-alkyl-RNA units, 2′-OMe-RNA units, 2′-amino-DNA units,and 2′-fluoro-DNA units.

In some embodiments, the nucleotide analogues comprise or are LockedNucleic Acid (LNA) units.

In some embodiments, the oligomer of the invention comprises or is agapmer, such as a LNA gapmer oligonucleotide.

In some embodiments, the Gapmer comprise a wing on each side (5′ and 3′)of 2 to 4 nucleotide analogues, preferably LNA analogues.

In some embodiments, the oligomer of the invention comprises acontiguous sequence of 13, 14, 15 or 16 nucleotides which arecomplementary to a corresponding length of SEQ ID NO 33 or 34 or 45 or acontiguous sequence of 16 nucleotides which are complementary to acorresponding length of SEQ ID NO 31, and may optionally comprise afurther 1-6 nucleotides, which may form or comprise a biocleavablenucleotide region, such as a phosphate nucleotide linker. Suitably, thebiocleavable nucleotide region is formed of a short stretch (eg. 1, 2,3, 4, 5 or 6) of nucleotides which are physiologically labile. This maybe achieved by using phosphodiester linkages with DNA/RNA nucleosides,or if physiological liability can be maintained, other nucleoside may beused. Physiological liability may be measured using a liver extract, asillustrated in example 6.

The oligomer of the invention may therefore comprise of a contiguousnucleotide sequence of 10-16 nts in length which is complementary to acorresponding length of SEQ ID NO 33 or 34 or 45 or a contiguoussequence of 16 nucleotides which are complementary to a correspondinglength of SEQ ID NO 31 (A first region, or region A). The oligomer ofthe invention may comprise a further nucleotide region. In someembodiments, the further nucleotide region comprises a biocleavablenucleotide region, such as a phosphate nucleotide sequence (a secondregion, region B), which may covalently link region A to anon-nucleotide moiety, such as a conjugate group, (a third region, orregion C). In some embodiments the contiguous nucleotide sequence of theoligomer of the invention (region A) is directly covalently linked toregion C. In some embodiments region C is biocleavable.

The oligomer consists or comprises of a contiguous nucleotide sequenceof from 12-22, such as 13, 14, 15, 16, 17, 18, 19, 20, 21, nucleotidesin length, such as 14-16 nucleotides in length, such as 15 or 16nucleotides in length. The oligomer may therefore refer to the combinedlength of region A and region B, e.g. (Region A 10-16 nt) and region B(1-6 nt).

In various embodiments, the compound of the invention does not compriseRNA (units). In some embodiments, the compound according to theinvention, the first region, or the first and second regions together(e.g. as a single contiguous sequence), is a linear molecule or issynthesised as a linear molecule. The oligomer may therefore be singlestranded molecule. In some embodiments, the oligomer does not compriseshort regions of, for example, at least 3, 4 or 5 contiguousnucleotides, which are complementary to equivalent regions within thesame oligomer (i.e. duplexes). The oligomer, in some embodiments, may benot (essentially) double stranded. In some embodiments, the oligomer isessentially not double stranded, such as is not a siRNA.

Oligomer Sequences

The following table provides oligomers and oligomer conjugates of theinvention and PCSK9 target sequences of the invention

TABLE 1 Position on the SEQ Chol- PCSK9 gene ID Sequence PO C6 GalNAcSEQ ID NO 44 1 TGCtacaaaacCCA 3643-3656 2 AATgctacaaaaCCCA 3643-3658 3AATgctacaaaacCCA 3643-3658 4 GCtgtgtgagcttGG 3251-3265 5TGctgtgtgagctTGG 3251-3266 6 TGCtgtgtgagctTGG 3251-3266 7TCCtggtctgtgtTCC 3373-3388 8 TCCtggtctgtgttCC 3373-3388 9 TGCtacaaaacCCAyes yes 3643-3656 10 AATgctacaaaaCCCA yes yes 3643-3658 11AATgctacaaaacCCA yes yes 3643-3658 12 GCtgtgtgagcttGG yes yes 3251-326513 TGctgtgtgagctTGG yes yes 3251-3266 14 TGCtgtgtgagctTGG yes yes3251-3266 15 TCCtggtctgtgtTCC yes yes 3373-3388 16 TCCtggtctgtgttCC yesyes 3373-3388 17 TGCtacaaaacCCA yes 3643-3656 18 AATgctacaaaaCCCA yes3643-3658 19 AATgctacaaaacCCA yes 3643-3658 20 GCtgtgtgagcttGG yes3251-3265 21 TGctgtgtgagctTGG yes 3251-3266 22 TGCtgtgtgagctTGG yes3251-3266 23 TCCtggtctgtgtTCC yes 3373-3388 24 TCCtggtctgtgttCC yes3373-3388 40 GTctgtggaaGCG 1005-1017 41 GTctgtggaaGCG yes 1005-1017 42GTctgtggaaGCG yes yes 1005-1017 43 GTctgtggaaGCG yes yes 1005-1017 25tgctacaaaaccca 3643-3656 26 aatgctacaaaaccca 3643-3658 27gctgtgtgagcttgg 3251-3265 28 tgctgtgtgagcttgg 3251-3266 29tcctggtctgtgttcc 3373-3388 44 gtctgtggaagcg 1005-1017 30 UGGGUUUUGUAGCA3643-3656 31 UGGGUUUUGUAGCAUU 3643-3658 32 CCAAGCUCACACAGC 3251-3265 33CCAAGCUCACACAGCA 3251-3266 34 GGAACACAGACCAGGA 3373-3388 45CGCUUCCACAGAC 1005-1017

SEQ ID NO 25-29 and 44 are nucleobase sequence motifs.

SEQ ID NOs 30-34 and 45 are the RNA target sequences present in thehuman PCSK9 mRNA.

SEQ ID NO 1 is SPC5001.

SEQ ID NOs 1-24 and 40 to 43 are oligomers comprising nucleotideanalogues such as LNA gapmer oligomers, where lower case letters are DNAunits (nucleoside/nucleotide) where capital letters are LNA units,

In some embodiments all LNA C are 5-methyl cytosine. In some embodimentsall LNA units are beta-D-oxy LNA. In some embodiments theinternucleoside linkages between the nucleosides of SEQ ID NOs 1-24 and40 to 43 are all phosphorothioate linkages.

SEQ ID NOs 9-16 and 41 to 43 comprise the oligomer (as indicated by theSEQ ID) as well as a cholesterol conjugate which may be covalentlylinked to the oligomer 5′ or 3′ end of the oligomer, optionally via abiocleavable linker, such as a phosphate nucleoside linker. In someembodiments, the cholesterol conjugate is linked at the 5′ end of theoligomer.

SEQ ID NOs 17-24 comprise the oligomer (as indicated by the SEQ ID) aswell as a GalNAc conjugate which may be covalently linked to theoligomer 5′ or 3′ end of the oligomer, optionally via a biocleavablelinker, such as a phosphate nucleoside linker or cleavable peptidelinker. In some embodiments, the GalNAc conjugate is linked at the 5′end of the oligomer.

Specific oligomers and conjugates used herein are illustrated in FIG. 3(oligomers), FIG. 4 (cholesterol conjugates), FIG. 5 (GalNAcconjugates). Other examples of conjugates which may be used with theoligomer of the invention are illustrated in FIGS. 1 and 2 and describedin section GalNAc Conjugate Moieties.

Table 2 provides specific combinations of oligomer and conjugates.

TABLE 2 Oligomer/conjugate combinations. SEQ Conjugate Number (SeeFIG. 1) ID Conj1 Conj2 Conj3 Conj4 Conj1a Conj2a Conj3a Conj4a 2 C1 C2C3 C4 C5 C6 C7 C8 3 C11 C12 C13 C14 C15 C16 C17 C18 4 C12 C13 C14 C15C16 C17 C18 C19 5 C30 C31 C32 C33 C34 C35 C36 C37 6 C40 C41 C42 C43 C44C45 C46 C47 7 C50 C51 C52 C53 C54 C55 C56 C57 8 C60 C61 C62 C63 C64 C65C66 C67 SEQ Conjugate Number (See FIG. 2) ID Conj5 Conj6 Conj5a Conj6a 2C9 C10 C70 C71 3 C19 C20 C72 C73 4 C20 C21 C74 C75 5 C38 C39 C76 C77 6C48 C49 C78 C79 7 C58 C59 C80 C81 8 C68 C69 C82 C83

All these combinations can be visualized by substituting the wavy linein FIG. 1 or 2 with the sequence of the oligomer. FIG. 5 show thecombination of Conj2a with the indicated SEQ ID NO's above. FIGS. 5A and5B are two detailed examples of the compounds in FIG. 5. Please notethat a biocleavable linker (B) may or may not be present between theconjugate moiety (C) and the oligomer (A). For Conj1-4 and 1a-4a theGalNAc conjugate itself is biocleavable, utilizing a peptide linker inthe GalNAc cluster, and as such a further biocleavable linker (B) may ormay not be used. However, preliminary data indicates inclusion of abiocleavable linker (B), such as the phosphate nucleotide linkersdisclosed herein may enhance activity of such GalNAc cluster oligomerconjugates. FIG. 4 shows the combination of Conj5a with the indicatedSEQ ID NO's above with a biocleavable linker (B) composed of two DNAmonomers C and A linked with a phosphodiester linkage. For use with Conj5 and Conj 6, the use of a biocleavable linker greatly enhances compoundactivity inclusion of a biocleavable linker (B), such as the phosphatenucleotide linkers disclosed herein is recommended.

The terms “corresponding to” and “corresponds to” refer to thecomparison between the nucleotide sequence of the oligomer (i.e. thenucleobase or base sequence) or contiguous nucleotide sequence (a firstregion/region A) and the reverse complement of the nucleic acid target,or sub-region thereof (e.g. SEQ ID NO 31, 32 33, 34 or 45). Nucleotideanalogues are compared directly to their equivalent or correspondingnucleotides. In a preferred embodiment, the oligomers (or first regionthereof) are complementary to the target region or sub-region thereof(e.g. SEQ ID NO 31, 32, 33, 34 or 45), such as fully complementary.

The terms “reverse complement”, “reverse complementary” and “reversecomplementarity” as used herein are interchangeable with the terms“complement”, “complementary” and “complementarity”.

The term, “complementary” means that two sequences are complementarywhen the sequence of one can bind to the sequence of the other in ananti-parallel sense wherein the 3′-end of each sequence binds to the5′-end of the other sequence and each A, T(U), G, and C of one sequenceis then aligned with a T(U), A, C, and G, respectively, of the othersequence. Normally, the complementary sequence of the oligonucleotidehas at least 90%, preferably 95%, most preferably 100%, complementarityto a defined sequence.

The terms “corresponding nucleotide analogue” and “correspondingnucleotide” are intended to indicate that the nucleotide in thenucleotide analogue and the naturally occurring nucleotide areidentical. For example, when the 2-deoxyribose unit of the nucleotide islinked to an adenine, the “corresponding nucleotide analogue” contains apentose unit (different from 2-deoxyribose) linked to an adenine.

The term “nucleobase” refers to the base moiety of a nucleotide andcovers both naturally occurring a well as non-naturally occurringvariants. Thus, “nucleobase” covers not only the known purine andpyrimidine heterocycles but also heterocyclic analogues and tautomeresthereof. It will be recognised that the DNA or RNA nucleosides of regionB may have a naturally occurring and/or non-naturally occurringnucleobase(s).

Examples of nucleobases include, but are not limited to adenine,guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine,5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,and 2-chloro-6-aminopurine. In some embodiments the nucleobases may beindependently selected from the group consisting of adenine, guanine,cytosine, thymidine, uracil, 5-methylcytosine. In some embodiments thenucleobases may be independently selected from the group consisting ofadenine, guanine, cytosine, thymidine, and 5-methylcytosine.

In some embodiments, at least one of the nucleobases present in theoligomer is a modified nucleobase selected from the group consisting of5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,and 2-chloro-6-aminopurine.

The Target

Suitably the oligomer of the invention is capable of modulating theexpression of the PCSK9 gene. Preferably the oligomer is capable ofdown-regulating expression of the PCSK9 gene. In this regards, theoligomer of the invention can affect the expression of PCSK9, typicallyin a mammalian such as a human cell, such as a liver cell. In someembodiments, the oligomers of the invention bind to the target nucleicacid and the effect on expression is at least 10% or 20% reductioncompared to the normal expression level (e.g. the expression level of acell, animal or human treated with saline), more preferably at least a30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition compared to thenormal expression level. In some embodiments, such modulation is seenwhen using between 0.04 and 25 nM, such as between 0.8 and 20 nMconcentration of the compound of the invention. In some embodiments,such modulation is seen when using between 0.01 and 15 mg/kg, such asbetween 0.05 and 10 mg/kg, such as between 0.1 and 7.5 mg/kg, such asbetween 0.25 and 5 mg/kg, such as 0.5 and 2.5 mg/kg concentration of thecompound of the invention. In the same or a different embodiment, theinhibition of expression is less than 100%, such as less than 98%inhibition, less than 95% inhibition, less than 90% inhibition, lessthan 80% inhibition, such as less than 70% inhibition. Modulation ofexpression level may be determined by measuring protein levels, e.g. bythe methods such as SDS-PAGE followed by western blotting using suitableantibodies raised against the target protein. Alternatively, modulationof expression levels can be determined by measuring levels of mRNA, e.g.by northern blotting or quantitative RT-PCR. When measuring via mRNAlevels, the level of down-regulation when using an appropriate dosage,such as between 0.04 and 25 nM, such as between 0.8 and 20 nMconcentration, is, in some embodiments, typically to a level of between10-20% the normal levels in the absence of the compound of theinvention.

The invention therefore provides a method of down-regulating orinhibiting the expression of PCSK9 protein and/or mRNA in a cell whichis expressing PCSK9 protein and/or mRNA, said method comprisingadministering the oligomer or conjugate according to the invention tosaid cell to down-regulating or inhibiting the expression of PCSK9protein and/or mRNA in said cell. Suitably the cell is a mammalian cellsuch as a human cell. The administration may occur, in some embodiments,in vitro. The administration may occur, in some embodiments, in vivo.

The term “target nucleic acid”, as used herein refers to the DNA or RNAencoding mammalian PCSK9 polypeptide, such as human PCSK9, such as NCBIaccession number NM_174936 SEQ ID NO: 46. PCSK9 encoding nucleic acidsor naturally occurring variants thereof, and RNA nucleic acids derivedtherefrom, preferably mRNA, such as pre-mRNA, although preferably maturemRNA. In some embodiments, for example when used in research ordiagnostics the “target nucleic acid” may be a cDNA or a syntheticoligonucleotide derived from the above DNA or RNA nucleic acid targets.The oligomer according to the invention is preferably capable ofhybridising to the target nucleic acid. It will be recognised that SEQID NO: 46 is a cDNA sequence, and as such, corresponds to the maturemRNA target sequence, although uracil is replaced with thymidine in thecDNA sequences.

The term “naturally occurring variant thereof” refers to variants of thePCSK9 polypeptide of nucleic acid sequence which exist naturally withinthe defined taxonomic group, such as mammalian, such as mouse, monkey,and preferably human. Typically, when referring to “naturally occurringvariants” of a polynucleotide the term also may encompass any allelicvariant of the PCSK9 encoding genomic DNA which are found at thechromosome 4, at 4 C7 by chromosomal translocation or duplication, andthe RNA, such as mRNA derived therefrom. “Naturally occurring variants”may also include variants derived from alternative splicing of the PCSK9mRNA. When referenced to a specific polypeptide sequence, e.g., the termalso includes naturally occurring forms of the protein which maytherefore be processed, e.g. by co- or post-translational modifications,such as signal peptide cleavage, proteolytic cleavage, glycosylation,etc.

In some embodiments the oligomer (or contiguous nucleotide portionthereof) is selected from, or comprises, one of the sequences selectedfrom the group consisting of SEQ ID NOS: 28 or 29 or 44, or asub-sequence of at least 10 contiguous nucleotides thereof, wherein saidoligomer (or contiguous nucleotide portion thereof) may optionallycomprise one, two, or three mismatches when compared to the sequence.

In some embodiments the target sequence is selected from, or comprisesor consists of, one of the sequences selected from the group consistingof SEQ ID NOs 31, 32, 33, 34 or 45, or a sub-sequence of at least 10contiguous nucleotides of SEQ ID NOs: 33, 34 or 45.

In some embodiments the sub-sequence may consist of 11, 12, 13, 14, 15or 16 contiguous nucleotides, such as between 12-16 nucleotides.Suitably, in some embodiments, the sub-sequence is of the same length asthe contiguous nucleotide sequence of the oligomer of the invention(optionally excluding region B when region B is not complementary to thetarget).

However, it is recognised that, in some embodiments the nucleotidesequence of the oligomer may comprise additional 5′ or 3′ nucleotides,such as, independently, 1, 2, 3, 4, 5 or 6 additional nucleotides 5′and/or 3′, which are non-complementary to the target sequence—suchnon-complementary oligonucleotides may form region B In this respect theoligomer of the invention, may, in some embodiments, comprise acontiguous nucleotide sequence which is flanked 5′ and or 3′ byadditional nucleotides. In some embodiments the additional 5′ or 3′nucleotides are naturally occurring nucleotides, such as DNA or RNA. Insome embodiments, the additional 5′ or 3′ nucleotides may representregion D as referred to in the context of gapmer oligomers herein.

In some embodiments, the oligomer according to the invention consists orcomprises of a nucleotide sequence according to SEQ ID NO: 27, or asub-sequence of at least 10 or 12 nucleobases thereof.

In some embodiments, the oligomer according to the invention consists orcomprises of a nucleotide sequence according to SEQ ID NO: 28, or asub-sequence of at least 10 or 12 nucleobases thereof. In a preferredembodiment, the oligomer according to the invention consists orcomprises of a nucleotide sequence according to SEQ ID NO: 5 or 6. Inanother preferred embodiment the oligomer conjugate according to theinvention consists or comprises of a nucleotide sequence according toSEQ ID NO: 13 or 14 or 21 or 22.

In some embodiments, the oligomer according to the invention consists orcomprises of a nucleotide sequence according to SEQ ID NO: 29, or asub-sequence of at least 10 or 12 nucleobases thereof. In a preferredembodiment, the oligomer according to the invention consists orcomprises of a nucleotide sequence according to SEQ ID NO: 7 or 8. Inanother preferred embodiment the oligomer conjugate according to theinvention consists or comprises of a nucleotide sequence according toSEQ ID NO: 15 or 16 or 23 or 24.

In some embodiments, the oligomer according to the invention consists orcomprises of a nucleotide sequence according to SEQ ID NO: 44, or asub-sequence of at least 10 or 12 nucleobases thereof. In a preferredembodiment, the oligomer according to the invention consists orcomprises of a nucleotide sequence according to SEQ ID NO: 40. Inanother preferred embodiment the oligomer conjugate according to theinvention consists or comprises of a nucleotide sequence according toSEQ ID NO: 41, 42 or 43.

In some embodiments the oligomer according to the invention consists orcomprises of a nucleotide sequence according to SEQ ID NO:26. In apreferred embodiment, the oligomer according to the invention consistsor comprises of a nucleotide sequence according to SEQ ID NO: 2 or 3. Inanother preferred embodiment, the oligomer conjugate according to theinvention consists or comprises of a nucleotide sequence according toSEQ ID NO: 10 or 11 or 18 or 19.

Length

The oligomers may comprise or consist of a contiguous nucleotidesequence of a total of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,or 22 contiguous nucleotides in length. Lengths may include region A orregion A and B for example.

In some embodiments, the oligomers comprise or consist of a contiguousnucleotide sequence of a total of between 10-22, such as 12-18, such as13-17 or 12-16, such as 13, 14, 15, 16 contiguous nucleotides in length.Preferably the oligomer of region A comprise or consist of a contiguousnucleotide sequence of 14 contiguous nucleotides in length, morepreferred of 15 contiguous nucleotides in length, and most preferred of16 contiguous nucleotides in length.

In some embodiments, the oligomer according to the invention consists ofno more than 22 nucleotides, such as no more than 20 nucleotides, suchas no more than 18 nucleotides, such as 15, 16 or 17 nucleotides. Insome embodiments, the oligomer of the invention comprises less than 20nucleotides.

Nucleotide Analogues

The term “nucleotide” as used herein, refers to a glycoside comprising asugar moiety, a base moiety and a covalently linked group, such as aphosphate or phosphorothioate internucleotide linkage group, and coversboth naturally occurring nucleotides, such as DNA or RNA, andnon-naturally occurring nucleotides comprising modified sugar and/orbase moieties, which are also referred to as “nucleotide analogues”herein. Herein, a single nucleotide (unit) may also be referred to as amonomer or nucleic acid unit.

In field of biochemistry, the term “nucleoside” is commonly used torefer to a glycoside comprising a sugar moiety and a base moiety. Thecovalent linkage between two nucleosides may be referred to as aninternucleoside linkage. Alternatively, the term internucleotide linkagemay be used to characterize the linkage between the nucleotides of theoligomer.

As one of ordinary skill in the art would recognise, the 5′ nucleotideof an oligonucleotide does not comprise a 5′ internucleotide linkagegroup, although may or may not comprise a 5′ terminal group, such as aphophodiester or phosphorothioate suitable for conjugating a linker (Bor Y or a conjugate moiety).

Non-naturally occurring nucleotides include nucleotides which havemodified sugar moieties, such as bicyclic nucleotides or 2′ modifiednucleotides, such as 2′ substituted nucleotides.

“Nucleotide analogues” are variants of natural nucleotides, such as DNAor RNA nucleotides, by virtue of modifications in the sugar and/or basemoieties. Analogues could in principle be merely “silent” or“equivalent” to the natural nucleotides in the context of theoligonucleotide, i.e. have no functional effect on the way theoligonucleotide works to inhibit target gene expression. Such“equivalent” analogues may nevertheless be useful if, for example, theyare easier or cheaper to manufacture, or are more stable to storage ormanufacturing conditions, or represent a tag or label. Preferably,however, the analogues will have a functional effect on the way in whichthe oligomer works to inhibit expression; for example by producingincreased binding affinity (affinity enhancing) to the target and/orincreased resistance to intracellular nucleases and/or increased ease oftransport into the cell. Specific examples of nucleoside analogues aredescribed by e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, andin Scheme 1:

The oligomer may thus comprise or consist of a simple sequence ofnatural occurring nucleotides—preferably 2′-deoxynucleotides (referredhere generally as “DNA”), but also possibly ribonucleotides (referredhere generally as “RNA”), or a combination of such naturally occurringnucleotides and one or more non-naturally occurring nucleotides, i.e.nucleotide analogues. Such nucleotide analogues may suitably enhance theaffinity of the oligomer for the target sequence. Examples of suitableand preferred nucleotide analogues are provided by WO2007/031091 or arereferenced therein.

Incorporation of affinity-enhancing nucleotide analogues in theoligomer, such as LNA or 2′-substituted sugars, can allow the size ofthe specifically binding oligomer to be reduced, and may also reduce theupper limit to the size of the oligomer before non-specific or aberrantbinding takes place.

In some embodiments the oligomer comprises at least 2 nucleotideanalogues. In some embodiments, the oligomer comprises from 3-8nucleotide analogues, e.g. 6 or 7 nucleotide analogues.

Examples of nucleotide analogues include modifying the sugar moiety toprovide a 2′-substituent group or to produce a bicyclic structure whichenhances binding affinity and may also provide increased nucleaseresistance.

In some embodiments, nucleotide analogues present within an antisenseoligomer of the present invention (such as in regions X′ and Y′mentioned in the section “Gapmer Design”) are independently selectedfrom, for example: 2′-O-alkyl-RNA units, 2′-OMe-RNA units,2′-O-alkyl-DNA, 2′-amino-DNA units, 2′-fluoro-DNA units, LNA units,arabino nucleic acid (ANA) units, 2′-fluoro-ANA units, HNA units, INA(intercalating nucleic acid—Christensen, 2002. Nucl. Acids. Res. 200230: 4918-4925, hereby incorporated by reference) units and 2′MOE units.

In some embodiments, nucleotide analogues are 2′-O-methoxyethyl-RNA(2′MOE), 2′-fluoro-DNA monomers or LNA nucleotide analogues, and as suchan antisense oligonucleotide of the present invention may comprisenucleotide analogues which are independently selected from these threetypes of analogue, or may comprise only one type of analogue selectedfrom the three types. In some embodiments at least one of saidnucleotide analogues is 2′-MOE-RNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 102′-MOE-RNA nucleotide units. In some embodiments, at least one of saidnucleotide analogues is 2′-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or10 2′-fluoro-DNA nucleotide units.

A preferred nucleotide analogue is LNA, such as oxy-LNA (such asbeta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA (such asbeta-D-amino-LNA and alpha-L-amino-LNA) and/or thio-LNA (such asbeta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENA andalpha-L-ENA). Most preferred is beta-D-oxy-LNA.

In some embodiments, there is only one of the above types of nucleotideanalogues present in an antisense oligonucleotide of the presentinvention, or contiguous nucleotide sequence thereof.

In some embodiments, an antisense oligonucleotide of the presentinvention comprises at least one Locked Nucleic Acid (LNA) unit, such as1, 2, 3, 4, 5, 6, 7, or 8 LNA units, such as from 3-7 or 4 to 8 LNAunits. In the by far most preferred embodiments, at least one of saidnucleotide analogues is a locked nucleic acid (LNA); for example atleast 3 or at least 4, or at least 5, or at least 6, or at least 7, or8, of the nucleotide analogues may be LNA. In some embodiments all thenucleotides analogues may be LNA.

In some embodiments, an antisense oligonucleotide of the presentinvention may comprise both nucleotide analogues (preferably LNA) andDNA units. Preferably, the combined total of nucleotide analogues(preferably LNA) and DNA units is 10-25, such as 10-24, preferably10-20, such as 10-18, even more preferably 12-16. In some embodiments,the nucleotide sequence of an antisense oligonucleotide of the presentinvention, such as the contiguous nucleotide sequence, consists of atleast one nucleotide analogue (preferably LNA) and the remainingnucleotide units are DNA units. In some embodiments, an antisenseoligonucleotide of the present invention comprises only LNA nucleotideanalogues and naturally occurring nucleotides (such as RNA or DNA, mostpreferably DNA nucleotides), optionally with modified internucleotidelinkages such as phosphorothioate.

It will be recognised that when referring to a preferred nucleotidesequence motif or nucleotide sequence, which consists of onlynucleotides, the oligomers of the invention which are defined by thatsequence may comprise a corresponding nucleotide analogue in place ofone or more of the nucleotides present in said sequence, such as LNAunits or other nucleotide analogues, which raise the duplexstability/T_(m) of the oligomer/target duplex (i.e. affinity enhancingnucleotide analogues).

T_(m) Assay:

The oligonucleotide: Oligonucleotide and RNA target (PO) duplexes arediluted to 3 mM in 500 ml RNase-free water and mixed with 500 ml2×T_(m)-buffer (200 mM NaCl, 0.2 mM EDTA, 20 mM Naphosphate, pH 7.0).The solution is heated to 95° C. for 3 min and then allowed to anneal inroom temperature for 30 min. The duplex melting temperatures (T_(m)) ismeasured on a Lambda 40 UV/VIS Spectrophotometer equipped with a Peltiertemperature programmer PTP6 using PE Templab software (Perkin Elmer).The temperature is ramped up from 20° C. to 95° C. and then down to 25°C., recording absorption at 260 nm. First derivative and the localmaximums of both the melting and annealing are used to assess the duplexT_(m).

In some embodiments, any mismatches between the nucleotide sequence ofthe oligomer and the target sequence are preferably found in regionsoutside the affinity enhancing nucleotide analogues, such as region Y′as referred to in the section “Gapmer Design, and/or at a position withnon-modified, such as DNA nucleotides, in the oligonucleotide, and/or inregions which are 5′ or 3′ to the contiguous nucleotide sequence.

LNA

The term “LNA” refers to a bicyclic nucleoside analogue which compriseswith a bridge between the 2′ and 4′ position in the ribose ring (2′ to4′ bicyclic nucleotide analogue), and is known as “Locked NucleicAcid”). LNA is in the literature sometimes referred to as BNA (bridgednucleic acid or bicyclic nucleic acid) and the two terms may be usedinterchangeably. The term LNA may refer to an LNA monomer, or, when usedin the context of an “LNA oligonucleotide”, LNA refers to anoligonucleotide containing one or more such bicyclic nucleotideanalogues. In some aspects bicyclic nucleoside analogues are LNAnucleotides, and these terms may therefore be used interchangeably, andis such embodiments, both are be characterized by the presence of alinker group (such as a bridge) between C2′ and C4′ of the ribose sugarring.

In some embodiments, an antisense oligonucleotide of the presentinvention may comprise both beta-D-oxy-LNA, and one or more of thefollowing LNA units: thio-LNA, amino-LNA, oxy-LNA, 5′-methyl-LNA and/orENA in either the beta-D or alpha-L configurations or combinationsthereof. In some embodiments, all LNA cytosine units are5′-methyl-Cytosine. In some embodiments, at least one nucleosideanalogue present in the first region (X′) is a bicyclic nucleosideanalogue, such as at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, (except the DNA and or RNA nucleosidesof region Y′) are sugar modified nucleoside analogues, such as such asbicyclic nucleoside analogues, such as LNA, e.g. beta-D-X-LNA oralpha-L-X-LNA (wherein X is oxy, amino or thio), or other LNAs disclosedherein including, but not limited to, (R/S) cET, cMOE or 5′-Me-LNA.

In some embodiments the LNA used in the oligonucleotide compounds of theinvention preferably has the structure of the general formula II:

wherein Y is selected from the group consisting of —O—, —CH₂O—, —S—,—NH—, N(R^(e)) and/or —CH₂—; Z and Z* are independently selected amongan internucleotide linkage, R^(H), a terminal group or a protectinggroup; B constitutes a natural or non-natural nucleotide base moiety(nucleobase), and R^(H) is selected from hydrogen and C₁₋₄-alkyl; R^(a),R^(b) R^(c), R^(d) and R^(e) are, optionally independently, selectedfrom the group consisting of hydrogen, optionally substitutedC₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl, optionallysubstituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl,C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl,formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂);and R^(H) is selected from hydrogen and C₁₋₄-alkyl. In some embodimentsR^(e), R^(b) R^(c), R^(d) and R^(e) are, optionally independently,selected from the group consisting of hydrogen and C₁₋₆alkyl, such asmethyl. For all chiral centers, asymmetric groups may be found in eitherR or S orientation, for example, two exemplary stereochemical isomersinclude the beta-D and alpha-L isoforms, which may be illustrated asfollows:

Specific exemplary LNA units are shown below:

The term “thio-LNA” comprises a locked nucleotide in which Y in thegeneral formula above is selected from S or —CH₂—S—. Thio-LNA can be inboth beta-D and alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which Y in thegeneral formula above is selected from —N(H)—, N(R)—, CH₂—N(H)—, and—CH₂—N(R)— where R is selected from hydrogen and C₁₋₄-alkyl. Amino-LNAcan be in both beta-D and alpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which Y in thegeneral formula above represents —O—. Oxy-LNA can be in both beta-D andalpha-L-configuration.

The term “ENA” comprises a locked nucleotide in which Y in the generalformula above is —CH₂—O— (where the oxygen atom of —CH₂—O— is attachedto the 2′-position relative to the base B). R^(e) is hydrogen or methyl.

In some exemplary embodiments LNA is selected from beta-D-oxy-LNA,alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particularbeta-D-oxy-LNA.

As used herein, “bicyclic nucleosides” refer to modified nucleosidescomprising a bicyclic sugar moiety. Examples of bicyclic nucleosidesinclude, without limitation, nucleosides comprising a bridge between the4′ and the 2′ ribosyl ring atoms. In some embodiments, compoundsprovided herein include one or more bicyclic nucleosides wherein thebridge comprises a 4′ to 2′ bicyclic nucleoside. Examples of such 4′ to2′ bicyclic nucleosides, include, but are not limited to, one of theformulae: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA);4′-CH(CH₃)—O-2′ and 4′-CH(CH₂OCH₃)—O-2*, and analogs thereof (see, U.S.Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′, andanalogs thereof (see, published PCT International ApplicationWO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′, and analogsthereof (see, published PCT International Application WO2008/150729,published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see, published U.S. PatentApplication US2004/0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′,wherein R is H, C₁-C₁₀ alkyl, or a protecting group (see, U.S. Pat. No.7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see,Chattopadhyaya, et al, J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′, and analogs thereof (see, published PCT InternationalApplication WO 2008/154401, published on Dec. 8, 2008). Also see, forexample: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett.,1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;Srivastava et al., J. Am. Chem. Soc, 129(26) 8362-8379 (Jul. 4, 2007);Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braaschet al., Chem. Biol, 2001, 8, 1-7; Oram et al, Curr. Opinion Mol. Ther.,2001, 3, 239-243; U.S. Pat. Nos. 6,670,461, 7,053,207, 6,268,490,6,770,748, 6,794,499, 7,034,133, 6,525,191, 7,399,845; published PCTInternational applications WO 2004/106356, WO 94/14226, WO 2005/021570,and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570,US2007/0287831, and US2008/0039618; and U.S. patent Ser. Nos.12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231,61/097,787, and 61/099,844; and PCT International Application Nos.PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922. Each of theforegoing bicyclic nucleosides can be prepared having one or morestereochemical sugar configurations including for examplea-L-ribofuranose and beta-D-ribofuranose (see PCT internationalapplication PCT DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

In some embodiments, bicyclic sugar moieties of LNA nucleosides include,but are not limited to, compounds having at least one bridge between the4′ and the 2′ position of the pentofuranosyl sugar moiety wherein suchbridges independently comprises 1 or from 2 to 4 linked groupsindependently selected from —[CiR_(a)XR_(b))], —, —C(R_(a))═C(R_(b))—,—C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—,—S(═O)_(x)—, and —N(Ra)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4;each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-Ci₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-Ci₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₆alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycleradical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl,substituted C₁-C₁₂ aminoalkyl, or a protecting group.

In some embodiments, the bridge of a bicyclic sugar moiety is,—[C(R_(a))(Rb)]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O—or, —C(R_(a)R_(b))—O—N(R)—. In some embodiments, the bridge is4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′, 4*—(CH₂)₂—O-2′,4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each R is,independently, H, a protecting group, or C₁-C₁₂ alkyl.

In some embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the a-L configuration or in the beta-Dconfiguration. Previously, a-L-methyleneoxy (4′-CH₂—O-2′) BNA's havebeen incorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al, Nucleic Acids Research, 2003, 21, 6365-6372).

In some embodiments, bicyclic nucleosides include, but are not limitedto, (A) a-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) beta-D-Methyleneoxy(4′-CH₂—O-2′) BNA, (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F),Methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA, (G) methylene-thio(4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methylcarbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, and (J) propylene carbocyclic(4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is the base moiety and R is, independently, H, a protectinggroup or C₁-C₂ alkyl.

In some embodiments, bicyclic nucleoside is defined by Formula I:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(Rc)-CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(Rc)-, —CH₂—N(Rc)-O—, or —N(Rc)-O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium.

In some embodiments, bicyclic nucleoside is defined by Formula II:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium; Z_(a) is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆alkenyl, substituted C₂-C₆ alkynyl, acyl, substituted acyl, substitutedamide, thiol, or substituted thio.

In some embodiments, each of the substituted groups is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), NJ_(d), SJ_(c), N₃, OC(═X)J_(c),and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d), and J_(e) is,independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl and X is O orNJ_(c).

In some embodiments, bicyclic nucleoside is defined by Formula III:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, orsubstituted acyl (C(═O)—).

In some embodiments, bicyclic nucleoside is defined by Formula IV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl;each q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂—Ce alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl, substituted Q-C₆alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl, or substituted C₁-C₆aminoalkyl;

In some embodiments, bicyclic nucleoside is defined by Formula V:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium; q_(a), q_(b), q_(c) and q_(f)are each, independently, hydrogen, halogen, C₁-C₁₂ alkyl, substitutedC₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, substituted C₁-C₁₂alkoxy, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, ON,C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k),N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k); orq_(e) and q_(f) together are ═C(q_(g))(q_(h)); q_(g) and q_(h) are each,independently, H, halogen, C₁-C₁₂ alkyl, or substituted C₁-C₁₂ alkyl.

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) BNAmonomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, anduracil, along with their oligomerization, and nucleic acid recognitionproperties have been described (see, e.g., Koshkin et al., Tetrahedron,1998, 54, 3607-3630). BNAs and preparation thereof are also described inWO 98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA, methyleneoxy (4′-CH₂—O-2′)BNA, and 2′-thio-BNAs, have also been prepared {see, e.g., Kumar et al.,Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of lockednucleoside analogs comprising oligodeoxyribonucleotide duplexes assubstrates for nucleic acid polymerases has also been described (see,e.g., Wengel et al., WO 99/14226). Furthermore, synthesis of2′-amino-BNA, a novel conformationally restricted high-affinityoligonucleotide analog, has been described in the art (see, e.g., Singhet al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino-and 2′-methylamino-BNA's have been prepared and the thermal stability oftheir duplexes with complementary RNA and DNA strands has beenpreviously reported.

In some embodiments, the bicyclic nucleoside is defined by Formula VI:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium; each qj, qj, q_(k) and ql is,independently, H, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl, substituted C₂-C₁₂ alkoxyl, OJ_(j),SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j),C(═O)NJ_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k),N(H)C(═O)NJ_(j)J_(k), or (H)C(═S)NJ_(j)J_(k); and qi and q_(j) or ql andq_(k) together are ═C(q_(g))(q_(h)), wherein q_(g) and q_(h) are each,independently, H, halogen, C₁-C₁₂ alkyl, or substituted C₁-C₆ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog, bridge 4′-CH═CH—CH₂-2′, have been described (see, e.g.,Freier et al, Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaeket al, J. Org. Chem., 2006, 71, 7731-77′40). The synthesis andpreparation of carbocyclic bicyclic nucleosides along with theiroligomerization and biochemical studies have also been described (see,e.g., Srivastava et al, J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclicnucleoside” refers to a bicyclic nucleoside comprising a furanose ringcomprising a bridge connecting the 2′ carbon atom and the 4′ carbonatom.

As used herein, “monocylic nucleosides” refer to nucleosides comprisingmodified sugar moieties that are not bicyclic sugar moieties. In someembodiments, the sugar moiety, or sugar moiety analogue, of a nucleosidemay be modified or substituted at any position.

As used herein, “2′-modified sugar” means a furanosyl sugar modified atthe 2′ position. In some embodiments, such modifications includesubstituents selected from: a halide, including, but not limited tosubstituted and unsubstituted alkoxy, substituted and unsubstitutedthioalkyl, substituted and unsubstituted amino alkyl, substituted andunsubstituted alkyl, substituted and unsubstituted allyl, andsubstituted and unsubstituted alkynyl. In some embodiments, 2′modifications are selected from substituents including, but not limitedto: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂), NH₂, O(CH₂), CH₃, O(CH₂), ONH₂,OCH₂C(═O)N(H)CH₃, and O(CH2)_(n)ON[(CH₂)_(n)CH₃]2, where n and m arefrom 1 to about 10. Other 2′-substituent groups can also be selectedfrom: C₁-C₁₂ alkyl; substituted alkyl; alkenyl; alkynyl; alkaryl;aralkyl; O-alkaryl or O-aralkyl; SH; SCH₃; OCN; Cl; Br; CN; CF₃; OCF₃;SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an R; a cleavinggroup; a reporter group; an intercalator; a group for improvingpharmacokinetic properties; and a group for improving thepharmacodynamic properties of an antisense compound, and othersubstituents having similar properties. In some embodiments, modifiednucleosides comprise a 2′-MOE side chain {see, e.g., Baker et al., J.Biol. Chem., 1997, 272, 1 1944-12000). Such 2′-MOE substitution havebeen described as having improved binding affinity compared tounmodified nucleosides and to other modified nucleosides, such as2′-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the2-MOE substituent also have been shown to be antisense inhibitors ofgene expression with promising features for in vivo use {see, e.g.,Martin, P., He/v. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia,1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24,630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16,917-926).

As used herein, a “modified tetrahydropyran nucleoside” or “modified THPnucleoside” means a nucleoside having a six-membered tetrahydropyran“sugar” substituted in for the pentofuranosyl residue in normalnucleosides (a sugar surrogate). Modified ?THP nucleosides include, butare not limited to, what is referred to in the art as hexitol nucleicacid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) {seeLeumann, CJ. Bioorg. and Med. Chem. (2002) 10:841-854), fluoro HNA(F-HNA), or those compounds defined by Formula X:

X wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the antisense compound and theother of T₃ and T4 is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′ or 3′-terminal group; q₁ q₂ q₃ q₄ q₅, q₆ and q₇ are each,independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆ alkynyl;and one of R₁ and R₂ is hydrogen and the other is selected from halogen,substituted or unsubstituted alkoxy, NJ, J₂, SJ, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X) NJ₁J₂, and CN, wherein X is O, S, or NJ₁ and eachJ₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In some embodiments, the modified THP nucleosides of Formula X areprovided wherein q_(m), q_(n), q_(p), q_(r), q_(s), q_(t), and q_(u) areeach H. In some embodiments, at least one of q_(m), q_(n), q_(p), q_(r),q_(s), q_(t) and q_(u) is other than H. In some embodiments, at leastone of q_(m), q_(n), q_(p), q_(r), q_(s), q_(t) and q_(u) is methyl. Insome embodiments, THP nucleosides of Formula X are provided wherein oneof R, and R₂ is F. In some embodiments, R₁ is fluoro and R₂ is H, R, ismethoxy and R₂ is H, and R, is methoxyethoxy and R₂ is H.

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides, include, but are not limited tonucleosides with non-bridging 2′ substituents, such as allyl, amino,azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R, is, independently,H or substituted or unsubstituted C₁-C₁₀ alkyl. 2′-modified nucleosidesmay further comprise other modifications, for example, at otherpositions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a sugar comprising a fluoro group atthe 2′ position.

As used herein, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” each refers to anucleoside comprising a sugar comprising an —OCH₃ group at the 2′position of the sugar ring.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides.

In some embodiments, one or more of the plurality of nucleosides ismodified. In some embodiments, an oligonucleotide comprises one or moreribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds {see, e.g., review article:Leumann, J. C, Bioorganic and Medicinal Chemistry, 2002, 10, 841-854).Such ring systems can undergo various additional substitutions toenhance activity. Methods for the preparations of modified sugars arewell known to those skilled in the art. In nucleotides having modifiedsugar moieties, the nucleobase moieties (natural, modified, or acombination thereof) are maintained for hybridization with anappropriate nucleic acid target.

In some embodiments, antisense compounds comprise one or morenucleotides having modified sugar moieties. In some embodiments, themodified sugar moiety is 2′-MOE. In some embodiments, the 2′-MOEmodified nucleotides are arranged in a gapmer motif. In someembodiments, the modified sugar moiety is a cEt. In some embodiments,the cEt modified nucleotides are arranged throughout the wings of agapmer motif.

In some embodiments, in the LNA, R⁴* and R²* together designate thebiradical —O—CH(CH₂OCH₃)— (2′O-methoxyethyl bicyclic nucleic acid—Sethat al., 2010, J. Org. Chem)—in either the R— or S— configuration.

In some embodiments, in the LNA, R⁴* and R²* together designate thebiradical —O—CH(CH₂CH₃)— (2′O-ethyl bicyclic nucleic acid—Seth at al.,2010, J. Org. Chem).—in either the R— or S— configuration.

In some embodiments, in the LNA, R⁴* and R²* together designate thebiradical —O—CH(CH₃)—. —in either the R— or S— configuration. In someembodiments, R⁴* and R²* together designate the biradical —O—CH₂—O—CH₂——(Seth at al., 2010, J. Org. Chem).

In some embodiments, in the LNA, R⁴* and R²* together designate thebiradical —O—NR—CH₃—(Seth at al., 2010, J. Org. Chem).

In some embodiments, the LNA units have a structure selected from thefollowing group:

Incorporation of affinity-enhancing nucleotide analogues in theoligomer, such as LNA or 2′-substituted sugars, can allow the size ofthe specifically binding oligomer to be reduced, and may also reduce theupper limit to the size of the oligomer before non-specific or aberrantbinding takes place.

We have evaluated the nephrotoxicity of a cET compound (using (S)-cET,with the sequence (Compound ID 6/411847 of WO2009/12495 and acomparative beta-D-oxy LNA compound (6/392063 of WO2009/12495) and foundthat the cET compounds elicit surprisingly high nephrotoxicity ascompared to the beta-D-oxy LNA control. The study was a single dosestudy, with sacrifice after 3 days (see EP1984381 example 41 for themethodology, although we used NMRI mice). Nephrotoxicity was confirmedby histological analysis. Notably signs of nephrotoxicity we seen atdosages of the cET compound below those where serum ALT was noted,indicating that for cET compounds, nephrotoxicity may be a particularproblem. The use of the conjugates of the present invention, such astrivalent GalNAc conjugates are therefore highly useful in reducing thenephrotoxicity of LNA compounds, such as cET compounds.

In some embodiments, the oligomer comprises at least 1 nucleosideanalogue. In some embodiments the oligomer comprises at least 2nucleotide analogues. In some embodiments, the oligomer comprises from3-8 nucleotide analogues, e.g. 6 or 7 nucleotide analogues. In the byfar most preferred embodiments, at least one of said nucleotideanalogues is a locked nucleic acid (LNA); for example at least 3 or atleast 4, or at least 5, or at least 6, or at least 7, or 8, of thenucleotide analogues may be LNA. In some embodiments all the nucleotidesanalogues may be LNA.

It will be recognized that when referring to a preferred nucleotidesequence motif or nucleotide sequence, which consists of onlynucleotides, the oligomers of the invention which are defined by thatsequence may comprise a corresponding nucleotide analogue in place ofone or more of the nucleotides present in said sequence, such as LNAunits or other nucleotide analogues, which raise the duplexstability/T_(m) of the oligomer/target duplex (i.e. affinity enhancingnucleotide analogues).

A preferred nucleotide analogue is LNA, such as oxy-LNA (such asbeta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA (such asbeta-D-amino-LNA and alpha-L-amino-LNA) and/or thio-LNA (such asbeta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENA andalpha-L-ENA).

In some embodiments, the oligomer of the invention, such as region A,may comprise LNA units and other nucleotide analogues, furthernucleotide analogues present within the oligomer of the invention areindependently selected from, for example: 2′-O-alkyl-RNA units,2′-amino-DNA units, 2′-fluoro-DNA units, LNA units, arabino nucleic acid(ANA) units, 2′-fluoro-ANA units, HNA units, INA (intercalating nucleicacid-Christensen, 2002. Nucl. Acids. Res. 2002 30: 4918-4925, herebyincorporated by reference) units and 2′MOE units. In some embodimentsthere is only one of the above types of nucleotide analogues present inthe oligomer of the invention, such as the first region, or contiguousnucleotide sequence thereof.

In some embodiments, the oligomer according to the invention (region A)may therefore comprises at least one Locked Nucleic Acid (LNA) unit,such as 1, 2, 3, 4, 5, 6, 7, or 8 LNA units, such as from 3-7 or 4 to 8LNA units, or 3, 4, 5, 6 or 7 LNA units. In some embodiments, all thenucleotide analogues are LNA. In some embodiments, the oligomer maycomprise both beta-D-oxy-LNA, and one or more of the following LNAunits: thio-LNA, amino-LNA, oxy-LNA, and/or ENA in either the beta-D oralpha-L configurations or combinations thereof. In some embodiments allLNA, cytosine units are 5′methyl-Cytosine. In some embodiments of theinvention, the oligomer (such as the first and optionally secondregions) may comprise both LNA and DNA units. In some embodiments, thecombined total of LNA and DNA units is 10-25, such as 10-24, preferably10-20, such as 10-18, such as 12-16. In some embodiments of theinvention, the nucleotide sequence of the oligomer, of first regionthereof, such as the contiguous nucleotide sequence consists of at leastone LNA and the remaining nucleotide units are DNA units. In someembodiments the oligomer, or first region thereof, comprises only LNA,nucleotide analogues and naturally occurring nucleotides (such as RNA orDNA, most preferably DNA nucleotides), optionally with modifiedinternucleotide linkages such as phosphorothioate.

RNAse Recruitment

It is recognized that an oligomeric compound may function via non RNasemediated degradation of target mRNA, such as by steric hindrance oftranslation, or other methods, In some embodiments, the oligomers of theinvention are capable of recruiting an endoribonuclease (RNase), such asRNase H.

It is preferable such oligomers, such as region A, or contiguousnucleotide sequence, comprises of a region of at least 4, such as atleast 5, such as at least 6, such as at least 7 consecutive nucleotideunits, such as at least 8 or at least 9 consecutive nucleotide units(residues), including 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutivenucleotides, which, when formed in a duplex with the complementarytarget RNA is capable of recruiting RNase (such as DNA units). Thecontiguous sequence which is capable of recruiting RNAse may be regionY′ as referred to in the context of a gapmer as described herein. Insome embodiments the size of the contiguous sequence which is capable ofrecruiting RNAse, such as region Y′, may be higher, such as 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 nucleotide units.

EP 1 222 309 provides in vitro methods for determining RNaseH activity,which may be used to determine the ability to recruit RNaseH. A oligomeris deemed capable of recruiting RNase H if, when provided with thecomplementary RNA target, it has an initial rate, as measured inpmol/l/min, of at least 1%, such as at least 5%, such as at least 10%or, more than 20% of the of the initial rate determined using DNA onlyoligonucleotide, having the same base sequence but containing only DNAmonomers, with no 2′ substitutions, with phosphorothioate linkage groupsbetween all monomers in the oligonucleotide, using the methodologyprovided by Example 91-95 of EP 1 222 309.

In some embodiments, an oligomer is deemed essentially incapable ofrecruiting RNaseH if, when provided with the complementary RNA target,and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is lessthan 1%, such as less than 5%, such as less than 10% or less than 20% ofthe initial rate determined using the equivalent DNA onlyoligonucleotide, with no 2′ substitutions, with phosphorothioate linkagegroups between all nucleotides in the oligonucleotide, using themethodology provided by Example 91-95 of EP 1 222 309.

In other embodiments, an oligomer is deemed capable of recruiting RNaseHif, when provided with the complementary RNA target, and RNaseH, theRNaseH initial rate, as measured in pmol/l/min, is at least 20%, such asat least 40%, such as at least 60%, such as at least 80% of the initialrate determined using the equivalent DNA only oligonucleotide, with no2′ substitutions, with phosphorothioate linkage groups between allnucleotides in the oligonucleotide, using the methodology provided byExample 91-95 of EP 1 222 309.

Typically the region of the oligomer which forms the consecutivenucleotide units which, when formed in a duplex with the complementarytarget RNA is capable of recruiting RNase consists of nucleotide unitswhich form a DNA/RNA like duplex with the RNA target. The oligomer ofthe invention, such as the first region, may comprise a nucleotidesequence which comprises both nucleotides and nucleotide analogues, andmay be e.g. in the form of a gapmer.

Gapmer Design

In some embodiments, the oligomer of the invention, such as the firstregion, comprises or is a gapmer. A gapmer oligomer is an oligomer whichcomprises a contiguous stretch of nucleotides which is capable ofrecruiting an RNAse, such as RNAseH, such as a region of at least 6 or 7DNA nucleotides, referred to herein in as region Y′ (Y′), wherein regionY′ is flanked both 5′ and 3′ by regions of affinity enhancing nucleotideanalogues, such as from 1-6 nucleotide analogues 5′ and 3′ to thecontiguous stretch of nucleotides which is capable of recruitingRNAse—these regions are referred to as regions X′ (X′) and Z′ (Z′),respectively. The X′ and Z′ regions can also be termed the wings of theGapmer. Examples of gapmers are disclosed in WO2004/046160,WO2008/113832, and WO2007/146511.

In some embodiments, the monomers which are capable of recruiting RNAseare selected from the group consisting of DNA monomers, alpha-L-LNAmonomers, C4′ alkylayted DNA monomers (see PCT/EP2009/050349 and Vesteret al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300, herebyincorporated by reference), and UNA (unlinked nucleic acid) nucleotides(see Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporatedby reference). UNA is unlocked nucleic acid, typically where the C2-C3C—C bond of the ribose has been removed, forming an unlocked “sugar”residue. Preferably the gapmer comprises a (poly)nucleotide sequence offormula (5′ to 3′), X′—Y′—Z′, wherein; region X′ (X′) (5′ region)consists or comprises of at least one nucleotide analogue, such as atleast one LNA unit, such as from 1-6 nucleotide analogues, such as LNAunits, and; region Y′ (Y′) consists or comprises of at least four or atleast five consecutive nucleotides which are capable of recruiting RNAse(when formed in a duplex with a complementary RNA molecule, such as themRNA target), such as DNA nucleotides, and; region Z′ (Z′) (3′region)consists or comprises of at least one nucleotide analogue, such as atleast one LNA unit, such as from 1-6 nucleotide analogues, such as LNAunits.

In some embodiments, region X′ consists of 1, 2, 3, 4, 5 or 6 nucleotideanalogues, such as LNA units, such as from 2-5 nucleotide analogues,such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or4 LNA units; and/or region Z consists of 1, 2, 3, 4, 5 or 6 nucleotideanalogues, such as. LNA units, such as from 2-5 nucleotide analogues,such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or4 LNA units.

In some embodiments Y′ consists or comprises of 4, 5, 6, 7, 8, 9, 10, 11or 12 consecutive nucleotides which are capable of recruiting RNAse, orfrom 4-12 or from 6-10, or from 7-9, such as 8 consecutive nucleotideswhich are capable of recruiting RNAse. In some embodiments region Y′consists or comprises at least one DNA nucleotide unit, such as 1-12 DNAunits, preferably from 4-12 DNA units, more preferably from 6-10 DNAunits, such as from 7-10 DNA units, most preferably 8, 9 or 10 DNAunits.

In some embodiments region X′ consist of 3 or 4 nucleotide analogues,such as LNA, region X′ consists of 7, 8, 9 or 10 DNA units, and regionZ′ consists of 3 or 4 nucleotide analogues, such as LNA. Such designsinclude (X′—Y′—Z′) 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3,3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3. In a preferred embodiment the gapmeris a 3-9-4 gapmer, even more preferred it is a 3-10-3 gapmer.

Further gapmer designs are disclosed in WO2004/046160, which is herebyincorporated by reference. WO2008/113832, which claims priority fromU.S. provisional application 60/977,409 hereby incorporated byreference, refers to ‘shortmer’ gapmer oligomers. In some embodiments,oligomers presented here may be such shortmer gapmers.

In some embodiments the oligomer, e.g. region X′, is consisting of acontiguous nucleotide sequence of a total of 10, 11, 12, 13 or 14nucleotide units, wherein the contiguous nucleotide sequence comprisesor is of formula (5′-3′), X′—Y′—Z′ wherein; X′ consists of 1, 2 or 3nucleotide analogue units, such as LNA units; Y′ consists of 7, 8 or 9contiguous nucleotide units which are capable of recruiting RNAse whenformed in a duplex with a complementary RNA molecule (such as a mRNAtarget); and Z′ consists of 1, 2 or 3 nucleotide analogue units, such asLNA units.

In some embodiments X′ consists of 1 LNA unit. In some embodiments X′consists of 2 LNA units. In some embodiments X′ consists of 3 LNA units.In some embodiments Z′ consists of 1 LNA units. In some embodiments Z′consists of 2 LNA units. In some embodiments Z′ consists of 3 LNA units.In some embodiments Y′ consists of 7 nucleotide units. In someembodiments Y′ consists of 8 nucleotide units. In some embodiments Y′consists of 9 nucleotide units. In certain embodiments, region Y′consists of 10 nucleoside monomers. In certain embodiments, region Y′consists or comprises 1-10 DNA monomers. In some embodiments Y′comprises of from 1-9 DNA units, such as 2, 3, 4, 5, 6, 7, 8 or 9 DNAunits. In some embodiments Y′ consists of DNA units. In some embodimentsY′ comprises of at least one LNA unit which is in the alpha-Lconfiguration, such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA units in thealpha-L-configuration. In some embodiments Y′ comprises of at least onealpha-L-oxy LNA unit or wherein all the LNA units in thealpha-L-configuration are alpha-L-oxy LNA units. In some embodiments thenumber of nucleotides present in X′—Y′—Z′ are selected from the groupconsisting of (nucleotide analogue units—region Y′— nucleotide analogueunits): 1-8-1, 1-8-2, 2-8-1, 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2,1-8-4, 2-8-4, or; 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 1-9-3,3-9-1, 4-9-1, 1-9-4, or; 1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1,2-10-3, 3-10-2. In some embodiments the number of nucleotides inX′—Y′—Z′ are selected from the group consisting of: 2-7-1, 1-7-2, 2-7-2,3-7-3, 2-7-3, 3-7-2, 3-7-4, and 4-7-3. In certain embodiments, each ofregions X′ and Y′ consists of three LNA monomers, and region Y′ consistsof 8 or 9 or 10 nucleoside monomers, preferably DNA monomers. In someembodiments both X′ and Z′ consists of two LNA units each, and Y′consists of 8 or 9 nucleotide units, preferably DNA units. In variousembodiments, other gapmer designs include those where regions X′ and/orZ′ consists of 3, 4, 5 or 6 nucleoside analogues, such as monomerscontaining a 2′-O-methoxyethyl-ribose sugar (2′-MOE) or monomerscontaining a 2′-fluoro-deoxyribose sugar, and region Y′ consists of 8,9, 10, 11 or 12 nucleosides, such as DNA monomers, where regionsX′—Y′—Z′ have 3-9-3, 3-10-3, 5-10-5 or 4-12-4 monomers. Further gapmerdesigns are disclosed in WO 2007/146511A2, hereby incorporated byreference.

LNA Gapmers: A LNA gapmer is a gapmer oligomer (region A) whichcomprises at least one LNA nucleotide. SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8and 40 are LNA gapmer oligomers. The oligomers with a contiguoussequence of 10-16 nucleotides which are complementary to a correspondinglength of SEQ ID NO 33 or 34 or 45 may also be gapmer oligomers such asLNA gapmers.

Internucleotide Linkages

The nucleoside monomers of the oligomers (e.g. first and second regions)described herein are coupled together via internucleoside linkagegroups. Suitably, each monomer is linked to the 3′ adjacent monomer viaa linkage group.

The person having ordinary skill in the art would understand that, inthe context of the present invention, the 5′ monomer at the end of anoligomer does not comprise a 5′ linkage group, although it may or maynot comprise a 5′ terminal group, or a linkage group for conjugation.

The terms “linkage group” or “internucleotide linkage” are intended tomean a group capable of covalently coupling together two nucleotides.Specific and preferred examples include phosphate groups andphosphorothioate groups. Internucleoside linkage may be usedinterchangeably with internucleotide linkage.

The nucleotides of the oligomer of the invention or contiguousnucleotides sequence thereof are coupled together via linkage groups.Suitably each nucleotide is linked to the 3′ adjacent nucleotide via alinkage group.

Suitable internucleotide linkages include those listed withinWO2007/031091, for example the internucleotide linkages listed on thefirst paragraph of page 34 of WO2007/031091 (hereby incorporated byreference). It is, in some embodiments, other than the phosphodiesterlinkage(s) of region B (where present), it is preferred to modify theinternucleotide linkage from its normal phosphodiester to one that ismore resistant to nuclease attack, such as phosphorothioate orboranophosphate—these two, being cleavable by RNase H, also allow thatroute of antisense inhibition in reducing the expression of the targetgene.

In some embodiments the oligomer of the present invention comprises oneor more nucleoside linkages selected from the group consisting ofphosphorothioate, phosphorodithioate and boranophosphate.

Suitable sulphur (S) containing internucleotide linkages as providedherein may be preferred, such as phosphorothioate or phosphodithioate.Phosphorothioate internucleotide linkages are also preferred,particularly for the first region, such as in gapmers, mixmers, antimirssplice switching oligomers, and totalmers.

The term ‘mixmer’ refers to oligomers which comprise both naturally andnon-naturally occurring nucleotides, where, as opposed to gapmers,tailmers, and headmers there is no contiguous sequence of more than 5,and in some embodiments no more than 4 consecutive, such as no more thanthree consecutive, naturally occurring nucleotides, such as DNA units

The term “totalmer” refers to a single stranded oligomer which onlycomprises non-naturally occurring nucleosides, such as sugar-modifiednucleoside analogues.

For gapmers, the internucleotide linkages in the oligomer may, forexample be phosphorothioate or boranophosphate so as to allow RNase Hcleavage of targeted RNA. Phosphorothioate is preferred, for improvednuclease resistance and other reasons, such as ease of manufacture.

In one aspect, with the exception of the phosphodiester linkage betweenthe first and second region, and optionally within region B, theremaining internucleoside linkages of the oligomer of the invention, thenucleotides and/or nucleotide analogues are linked to each other bymeans of phosphorothioate groups. In some embodiments, at least 50%,such as at least 70%, such as at least 80%, such as at least 90% such asall the internucleoside linkages between nucleosides in the first regionare other than phosphodiester (phosphate), such as are selected from thegroup consisting of phosphorothioate phosphorodithioate, orboranophosphate. In some embodiments, at least 50%, such as at least70%, such as at least 80%, such as at least 90% such as all theinternucleoside linkages between nucleosides in the first region arephosphorothioate.

WO09124238 refers to oligomeric compounds having at least one bicyclicnucleoside attached to the 3′ or 5′ termini by a neutral internucleosidelinkage. The oligomers of the invention may therefore have at least onebicyclic nucleoside attached to the 3′ or 5′ termini by a neutralinternucleoside linkage, such as one or more phosphotriester,methylphosphonate, MMI, amide-3, formacetal or thioformacetal. Theremaining linkages may be phosphorothioate.

Oligomer Conjugates (Region C)

A further aspect of the invention is an antisense oligonucleotideconjugate comprising an oligomer of the invention, and at least onenon-nucleotide or non-polynucleotide moiety (C) covalently attached tosaid oligomer (A), optionally via a linker region positioned between thecontiguous sequence of the oligomer and the conjugate moiety (B and/orY).

Representative conjugate moieties which have been used witholigonucleotides can include lipophilic molecules (aromatic andnon-aromatic) including steroid molecules; proteins (e.g., antibodies,enzymes, serum proteins); peptides; vitamins (water-soluble orlipid-soluble); polymers (water-soluble or lipid-soluble); smallmolecules including drugs, toxins, reporter molecules, and receptorligands; carbohydrate complexes; nucleic acid cleaving complexes; metalchelators (e.g., porphyrins, texaphyrins, crown ethers, etc.);intercalators including hybrid photonuclease/intercalators; crosslinkingagents (e.g., photoactive, redox active), and combinations andderivatives thereof. Numerous suitable conjugate moieties, theirpreparation and linkage to oligomeric compounds are provided, forexample, in WO 93/07883 and U.S. Pat. No. 6,395,492, each of which isincorporated herein by reference in its entirety. Oligonucleotideconjugates and their syntheses are also reported in comprehensivereviews by Manoharan in Antisense Drug Technology, Principles,Strategies, and Applications, S. T. Crooke, ed., Ch. 16, Marcel Dekker,Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development,2002, 12, 103, each of which is incorporated herein by reference in itsentirety.

In some embodiments the oligomer of the invention is targeted to theliver—i.e. after systemic administration the compound accumulates in theliver cells (such as hepatocytes). Targeting to the liver can be greatlyenhanced by the addition of a conjugate moiety (C). However, in order tomaximize the efficacy of the oligomer it is often desirable that theconjugate (or targeting moiety) is linked to the oligomer via abiocleavable linker (B), such as a nucleotide phosphate linker. It istherefore desirable to use a conjugate moiety which enhances uptake andactivity in hepatocytes. The enhancement of activity may be due toenhanced uptake or it may be due to enhanced potency of the compound inhepatocytes.

In some embodiments, the oligomeric compound is a LNA oligomer, such asa gapmer, or for example an LNA antisense oligomer, (which may bereferred to as region A herein) comprising an antisense oligomer,optionally a biocleavable linker, such as region B, and a carbohydrateconjugate (which may be referred to as region C). The LNA antisenseoligomer may be 7-30, such as 8-26 nucleosides in length and itcomprises at least one LNA unit (nucleoside).

In some embodiments, the conjugate is or may comprise a carbohydrate orcomprises a carbohydrate group. In some embodiments, the carbohydrate isselected from the group consisting of galactose, lactose,n-acetylgalactosamine, mannose, and mannose-6-phosphate. In someembodiments, the conjugate group is or may comprise mannose ormannose-6-phosphate. Carbohydrate conjugates may be used to enhancedelivery or activity in a range of tissues, such as liver and/or muscle.See, for example, EP1495769, WO99/65925, Yang et al., Bioconjug Chem(2009) 20(2): 213-21. Zatsepin & Oretskaya Chem Biodivers. (2004) 1(10):1401-17.

In some embodiments the carbohydrate moiety is not a linear carbohydratepolymer. In some embodiments, the oligomeric compound is a LNA oligomer,for example an LNA antisense oligomer, (which may be referred to asregion A herein) comprising an antisense oligomer, region B as definedherein, and an asialoglycoprotein receptor targeting moiety conjugatemoiety, such as a GalNAc moiety (which may be referred to as region C).The carbohydrate moiety may be multi-valent, such as, for example 2, 3,4 or 4 identical or non-identical carbohydrate moieties may becovalently joined to the oligomer, optionally via a linker or linkers(such as region Y).

GalNAc Conjugate Moieties

In some embodiments the carbohydrate moiety is not a linear carbohydratepolymer. The carbohydrate moiety may however be multi-valent, such as,for example 2, 3, 4 or 4 identical or non-identical carbohydratemoieties may be covalently joined to the oligomer, optionally via alinker or linkers. In some embodiments the invention provides aconjugate comprising the oligomer of the invention and a carbohydrateconjugate moiety. In some embodiments the invention provides a conjugatecomprising the oligomer of the invention and an asialoglycoproteinreceptor targeting moiety conjugate moiety, such as a GalNAc moiety,which may form part of a further region (referred to as region C).

The invention also provides LNA antisense oligonucleotides which areconjugated to an asialoglycoprotein receptor targeting moiety. In someembodiments, the conjugate moiety (such as the third region or region C)comprises an asialoglycoprotein receptor targeting moiety, such asgalactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine,N-propionyl-galactosamine, N-n-butanoyl-galactosamine, andN-isobutanoylgalactos-amine. In some embodiments the conjugate comprisesa galactose cluster, such as N-acetylgalactosamine trimer. In someembodiments, the conjugate moiety comprises an GalNAc(N-acetylgalactosamine), such as a mono-valent, di-valent, tri-valent oftetra-valent GalNAc. Trivalent GalNAc conjugates may be used to targetthe compound to the liver. GalNAc conjugates have been used withmethylphosphonate and PNA antisense oligonucleotides (e.g. U.S. Pat. No.5,994,517 and Hangeland et al., Bioconjug Chem. 1995 November-December;6(6):695-701) and siRNAs (e.g. WO2009/126933, WO2012/089352 &WO2012/083046). The GalNAc references and the specific conjugates usedtherein are hereby incorporated by reference. WO2012/083046 disclosessiRNAs with GalNAc conjugate moieties which comprise cleavablepharmacokinetic modulators, which are suitable for use in the presentinvention, the preferred pharmacokinetic modulators are C16 hydrophobicgroups such as palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl. The '046cleavable pharmacokinetic modulators may also be cholesterol.

The ‘targeting moieties (conjugate moieties) may be selected from thegroup consisting of: galactose, galactosamine, N-formyl-galactosamine,N-acetylgalactosamine, N-propionyl-galactosamine,N-n-butanoyl-galactosamine, N-iso-butanoylgalactos-amine, galactosecluster, and N-acetylgalactosamine trimer and may have a pharmacokineticmodulator selected from the group consisting of: hydrophobic grouphaving 16 or more carbon atoms, hydrophobic group having 16-20 carbonatoms, palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12dienoyl,dioctanoyl, and C16-C20 acyl, and cholesterol. Certain GalNAc clustersdisclosed in '046 include: (E)-hexadec-8-enoyl (C16), oleyl (C18),(9,E,12E)-octadeca-9,12-dienoyl (C18), octanoyl (C8), dodececanoyl(C12), C-20 acyl, C24 acyl, dioctanoyl (2×C8). The targetingmoiety-pharmacokinetic modulator targeting moiety may be linked to thepolynucleotide via a physiologically labile bond or, e.g. a disulfidebond, or a PEG linker. The invention also relates to the use ofphospodiester linkers between the oligomer and the conjugate group(these are referred to as region B herein, and suitably are positionedbetween the LNA oligomer and the carbohydrate conjugate group).

For targeting hepatocytes in liver, a preferred targeting ligand is agalactose cluster.

A galactose cluster comprises a molecule having e.g. comprising two tofour terminal galactose derivatives. As used herein, the term galactosederivative includes both galactose and derivatives of galactose havingaffinity for the asialoglycoprotein receptor equal to or greater thanthat of galactose. A terminal galactose derivative is attached to amolecule through its C—I carbon. The asialoglycoprotein receptor (ASGPr)is primarily expressed on hepatocytes and binds branchedgalactose-terminal glycoproteins. A preferred galactose cluster hasthree terminal galactosamines or galactosamine derivatives each havingaffinity for the asialoglycoprotein receptor. A more preferred galactosecluster has three terminal N-acetyl-galactosamines. Other terms commonin the art include tri-antennary galactose, tri-valent galactose andgalactose trimer. It is known that tri-antennary galactose derivativeclusters are bound to the ASGPr with greater affinity than bi-antennaryor mono-antennary galactose derivative structures (Baenziger and Fiete,1980, Cell, 22, 611-620; Connolly et al., 1982, 1. Biol. Chern.,257,939-945). Multivalency is required to achieve nM affinity. Accordingto WO 2012/083046 the attachment of a single galactose derivative havingaffinity for the asialoglycoprotein receptor does not enable functionaldelivery of the RNAi polynucleotide to hepatocytes in vivo whenco-administered with the delivery polymer.

A galactose cluster may comprise two or preferably three galactosederivatives each linked to a central branch point. The galactosederivatives are attached to the central branch point through the C—Icarbons of the saccharides. The galactose derivative is preferablylinked to the branch point via linkers or spacers. A preferred spacer isa flexible hydrophilic spacer (U.S. Pat. No. 5,885,968; Biessen et al.J. Med. Chern. 1995 Vol. 39 p. 1538-1546). A preferred flexiblehydrophilic spacer is a PEG spacer. A preferred PEG spacer is a PEG3spacer. The branch point can be any small molecule which permitsattachment of the three galactose derivatives and further permitsattachment of the branch point to the oligomer. An exemplary branchpoint group is a di-lysine. A di-lysine molecule contains three aminegroups through which three galactose derivatives may be attached and acarboxyl reactive group through which the di lysine may be attached tothe oligomer. Attachment of the branch point to oligomer may occurthrough a linker or spacer. A preferred spacer is a flexible hydrophilicspacer. A preferred flexible hydrophilic spacer is a PEG spacer. Apreferred PEG spacer is a PEG3 spacer (three ethylene units). Thegalactose cluster may be attached to the 3′ or 5′ end of the oligomerusing methods known in the art.

A preferred galactose derivative is an N-acetyl-galactosamine (GalNAc).Other saccharides having affinity for the asialoglycoprotein receptormay be selected from the list comprising: galactosamine,N-n-butanoylgalactosamine, and N-iso-butanoylgalactosamine. Theaffinities of numerous galactose derivatives for the asialoglycoproteinreceptor have been studied (see for example: Jobst, S. T. and Drickamer,K. JB. C. 1996,271,6686) or are readily determined using methods typicalin the art.

Further Examples of the conjugate of the invention are illustratedbelow:

Where at the hydrophobic or lipophilic (or further conjugate) moiety(i.e. pharmacokinetic modulator) in the above GalNAc cluster conjugatesis, when using LNA oligomers, such as LNA antisense oligonucleotides,optional.

See the figures for specific GalNAc clusters used in the present study,Conj 1, 2, 3, 4 and Conj1a, 2a, 3a and 4a (which are shown with anoptional C₆ linker which joins the GalNAc cluster to the oligomer).

In a preferred embodiment of the invention the conjugate moiety of theantisense oligonucleotide conjugate comprises or consists of Conj 1, 2,3, 4 and Conj1a, 2a, 3a and 4a. Most preferably the conjugate moietycomprises or consists of Conj 2a.

In another preferred embodiment the antisense oligonucleotide conjugateis selected from the group consisting of SEQ ID NO 17, 18, 19, 20, 21,22, 23, and 24.

Each carbohydrate moiety of a GalNAc cluster (e.g. GalNAc) may thereforebe joined to the oligomer via a spacer, such as (poly)ethylene glycollinker (PEG), such as a di, tri, tetra, penta, hexa-ethylene glycollinker. As is shown above the PEG moiety forms a spacer between thegalctose sugar moiety and a peptide (trilysine is shown) linker.

In some embodiments, the GalNAc cluster comprises a peptide linker, e.g.a Tyr-Asp(Asp) tripeptide or Asp(Asp) dipeptide, which is attached tothe oligomer (or to region Y or region B) via a biradical linker, forexample the GalNAc cluster may comprise the following biradical linkers:

R¹ is a biradical preferably selected from —C₂H₄—, —C₃H₆—, —C₄H₈—,—C₅H₁₀—, —C₆H₁₂—, 1,4-cyclohexyl (—C₆H₁₀—), 1,4-phenyl (—C₆H₄—),—O₂H₄OC₂H₄—, —C₂H₄(OC₂H₄)₂— or —C₂H₄(OC₂H₄)₃—, —C(O)CH₂—, —C(O)C₂H₄—,—C(O)C₃H₆—, —C(O)C₄H₈—, —C(O)C₅H₁₀—, —C(O)C₆H₁₂—, 1,4-cyclohexyl(—C(O)C₆H₁₀—), 1,4-phenyl (—C(O)C₆H₄—), —C(O)C₂H₄OC₂H₄—,—C(O)C₂H₄(OC₂H₄)₂— or —C(O)C₂H₄(OC₂H₄)₃—.

In some embodiments, R¹ is a biradical preferably selected from —C₂H₄—,—C₃H₆—, —C₄H₈—, —C₅H₁₀—, —C₆H₁₂—, 1,4-cyclohexyl (—C₆H₁₀-), 1,4-phenyl(—C₆H₄—), —C₂H₄OC₂H₄—, —C₂H₄(OC₂H₄)₂— or —C₂H₄(OC₂H₄)₃—.

The carbohydrate conjugate (e.g. GalNAc), or carbohydrate-linker moiety(e.g. carbohydrate-PEG moiety) may be covalently joined (linked) to theoligomer via a branch point group such as, an amino acid, or peptide,which suitably comprises two or more amino groups (such as 3, 4, or 5),such as lysine, di-lysine or tri-lysine or tetra-lysine. A tri-lysinemolecule contains four amine groups through which three carbohydrateconjugate groups, such as galactose & derivatives (e.g. GalNAc) and afurther conjugate such as a hydrophobic or lipophilic moiety/group maybe attached and a carboxyl reactive group through which the tri-lysinemay be attached to the oligomer. The further conjugate, such aslipophilic/hyrodphobic moiety may be attached to the lysine residue thatis attached to the oligomer.

Surprisingly, the present inventors have found that GalNAc conjugatesfor use with LNA oligomers do not require a pharmacokinetic modulator(as described below), and as such, in some embodiments, the GalNAcconjugate is not covalently linked to a lipophilic or hydrophobicmoiety, such as those described here in, e.g. do not comprise a C8-C36fatty acid or a sterol. The invention therefore also provides for LNAoligomer GalNAc conjugates which do not comprise a lipophilic orhydrophobic pharmacokinetic modulator or conjugate moiety/group.

Pharmacokinetic Modulators

The compound of the invention may further comprise one or moreadditional conjugate moieties, of which lipophilic or hydrophobicmoieties are particularly interesting, such as when the conjugate groupis a carbohydrate moiety. Such lipophilic or hydrophobic moieties mayact as pharmacokinetic modulators, and may be covalently linked toeither the carbohydrate conjugate, a linker linking the carbohydrateconjugate to the oligomer or a linker linking multiple carbohydrateconjugates (multi-valent) conjugates, or to the oligomer, optionally viaa linker, such as a bio cleavable linker.

The oligomer or conjugate moiety may therefore comprise apharmacokinetic modulator, such as a lipophilic or hydrophobic moieties.Such moieties are disclosed within the context of siRNA conjugates inWO2012/082046. The hydrophobic moiety may comprise a C8-C36 fatty acid,which may be saturated or un-saturated. In some embodiments, C10, C12,C14, C16, C18, C20, C22, C24, C26, C28, C30, C32 and C34 fatty acids maybe used. The hydrophobic group may have 16 or more carbon atoms.Exemplary suitable hydrophobic groups may be selected from the groupcomprising:sterol, cholesterol, palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl. According toWO '346, hydrophobic groups having fewer than 16 carbon atoms are lesseffective in enhancing polynucleotide targeting, but they may be used inmultiple copies (e.g. 2×, such as 2×C8 or C10, C12 or C14) to enhanceefficacy. Pharmacokinetic modulators useful as polynucleotide targetingmoieties may be selected from the group consisting of: cholesterol,alkyl group, alkenyl group, alkynyl group, aryl group, aralkyl group,aralkenyl group, and aralkynyl group, each of which may be linear,branched, or cyclic. Pharmacokinetic modulators are preferablyhydrocarbons, containing only carbon and hydrogen atoms. However,substitutions or heteroatoms which maintain hydrophobicity, for examplefluorine, may be permitted.

Lipophilic Conjugates

In some embodiments, the conjugate group is or may comprise a lipophilicmoiety, such as a sterol (for example, cholesterol, cholesteryl,cholestanol, stigmasterol, cholanic acid and ergosterol). In someembodiments the conjugate is or comprises tocopherol (exemplified asConj 6 and Conj 6a in FIG. 2). In some embodiments, the conjugate is ormay comprise cholesterol (exemplified as Conj 5 and Conj 5a in FIG. 2).

In some embodiments, the conjugate is, or may comprise a lipid, aphospholipid or a lipophilic alcohol, such as a cationic lipid, aneutral lipid, sphingolipid, and fatty acid such as stearic, oleic,elaidic, linoleic, linoleaidic, linolenic, and myristic acid. In someembodiments the fatty acid comprises a C4-C30 saturated or unsaturatedalkyl chain. The alkyl chain may be linear or branched.

Lipophilic conjugate moieties can be used, for example, to counter thehydrophilic nature of an oligomeric compound and enhance cellularpenetration.

Lipophilic moieties include, for example, sterols stanols, and steroidsand related compounds such as cholesterol (U.S. Pat. No. 4,958,013 andLetsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553),thiocholesterol (Oberhauser et al, Nucl Acids Res., 1992, 20, 533),lanosterol, coprostanol, stigmasterol, ergosterol, calciferol, cholicacid, deoxycholic acid, estrone, estradiol, estratriol, progesterone,stilbestrol, testosterone, androsterone, deoxycorticosterone, cortisone,17-hydroxycorticosterone, their derivatives, and the like. In someembodiments, the conjugate may be selected from the group consisting ofcholesterol, thiocholesterol, lanosterol, coprostanol, stigmasterol,ergosterol, calciferol, cholic acid, deoxycholic acid, estrone,estradiol, estratriol, progesterone, stilbestrol, testosterone,androsterone, deoxycorticosterone, cortisone, and17-hydroxycorticosterone. Other lipophilic conjugate moieties includealiphatic groups, such as, for example, straight chain, branched, andcyclic alkyls, alkenyls, and alkynyls. The aliphatic groups can have,for example, 5 to about 50, 6 to about 50, 8 to about 50, or 10 to about50 carbon atoms. Example aliphatic groups include undecyl, dodecyl,hexadecyl, heptadecyl, octadecyl, nonadecyl, terpenes, bornyl,adamantyl, derivatives thereof and the like. In some embodiments, one ormore carbon atoms in the aliphatic group can be replaced by a heteroatomsuch as O, S, or N (e.g., geranyloxyhexyl). Further suitable lipophilicconjugate moieties include aliphatic derivatives of glycerols such asalkylglycerols, bis(alkyl)glycerols, tris(alkyl)glycerols,monoglycerides, diglycerides, and triglycerides. In some embodiments,the lipophilic conjugate is di-hexyldecyl-rac-glycerol or 1,2-di-O—hexyldecyl-rac-glycerol (Manoharan et al., Tetrahedron Lett., 1995, 36,3651; Shea, et al., Nuc. Acids Res., 1990, 18, 3777) or phosphonatesthereof. Saturated and unsaturated fatty functionalities, such as, forexample, fatty acids, fatty alcohols, fatty esters, and fatty amines,can also serve as lipophilic conjugate moieties. In some embodiments,the fatty functionalities can contain from about 6 carbons to about 30or about 8 to about 22 carbons. Example fatty acids include, capric,caprylic, lauric, palmitic, myristic, stearic, oleic, linoleic,linolenic, arachidonic, eicosenoic acids and the like.

In further embodiments, lipophilic conjugate groups can be polycyclicaromatic groups having from 6 to about 50, 10 to about 50, or 14 toabout 40 carbon atoms. Example polycyclic aromatic groups includepyrenes, purines, acridines, xanthenes, fluorenes, phenanthrenes,anthracenes, quinolines, isoquinolines, naphthalenes, derivativesthereof and the like. Other suitable lipophilic conjugate moietiesinclude menthols, trityls (e.g., dimethoxytrityl (DMT)), phenoxazines,lipoic acid, phospholipids, ethers, thioethers (e.g.,hexyl-S-tritylthiol), derivatives thereof and the like. Preparation oflipophilic conjugates of oligomeric compounds are well-described in theart, such as in, for example, Saison-Behmoaras et al, EMBO J., 1991, 10,1111; Kabanov et al., FEBSLett., 1990, 259, 327; Svinarchuk et al,Biochimie, 1993, 75, 49; (Mishra et al., Biochim. Biophys. Acta, 1995,1264, 229, and Manoharan et al., Tetrahedron Lett., 1995, 36, 3651.

Oligomeric compounds containing conjugate moieties with affinity for lowdensity lipoprotein (LDL) can help provide an effective targeteddelivery system. High expression levels of receptors for LDL on tumorcells makes LDL an attractive carrier for selective delivery of drugs tothese cells (Rump, et al., Bioconjugate Chem., 1998, 9, 341; Firestone,Bioconjugate Chem., 1994, 5, 105; Mishra, et al., Biochim. Biophys.Acta, 1995, 1264, 229). Moieties having affinity for LDL include manylipophilic groups such as steroids (e.g., cholesterol), fatty acids,derivatives thereof and combinations thereof. In some embodiments,conjugate moieties having LDL affinity can be dioleyl esters of cholicacids such as chenodeoxycholic acid and lithocholic acid.

In some embodiments, the lipophilic conjugates may be or may comprisebiotin. In some embodiments, the lipophilic conjugate may be or maycomprise a glyceride or glyceride ester.

Lipophillic conjugates, such as sterols, stanols, and stains, such ascholesterol or as disclosed herein, may be used to enhance delivery ofthe oligonucleotide to, for example, the liver (typically hepatocytes).

In a preferred embodiment of the invention the conjugate moiety of theantisense oligonucleotide conjugate comprises or consists of Conj 5, 5a,6 or 6a. Most preferably the conjugate moiety comprises or consists ofConj 5a.

In another preferred embodiment the antisense oligonucleotide conjugateis selected from the group consisting of SEQ ID NO 9, 10, 11, 12, 13,14, 15, 16, 41, 42 and 43.

The following references also refer to the use of lipophilic conjugates:Kobylanska et al., Acta Biochim Pol. (1999); 46(3): 679-91. Felber etal., Biomaterials (2012) 33(25): 599-65); Grijalvo et al., J Org Chem(2010) 75(20): 6806-13. Koufaki et al., Curr Med Chem (2009) 16(35):4728-42. Godeau et al J. Med. Chem. (2008) 51(15): 4374-6.

Linkers (e.g. Region B or Y)

A linkage or linker is a connection between two atoms that links onechemical group or segment of interest to another chemical group orsegment of interest via one or more covalent bonds. Conjugate moieties(or targeting or blocking moieties) can be attached to the oligomericcompound directly or through a linking moiety (linker or tether)—alinker. Linkers are bifunctional moieties that serve to covalentlyconnect a third region, e.g. a conjugate moiety, to an oligomericcompound (such as to region A). In some embodiments, the linkercomprises a chain structure or an oligomer of repeating units such asethylene glyol or amino acid units. The linker can have at least twofunctionalities, one for attaching to the oligomeric compound and theother for attaching to the conjugate moiety. Example linkerfunctionalities can be electrophilic for reacting with nucleophilicgroups on the oligomer or conjugate moiety, or nucleophilic for reactingwith electrophilic groups. In some embodiments, linker functionalitiesinclude amino, hydroxyl, carboxylic acid, thiol, phosphoramidate,phosphorothioate, phosphate, phosphite, unsaturations (e.g., double ortriple bonds), and the like. Some example linkers include8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 6-aminohexanoicacid (AHEX or AHA), 6-aminohexyloxy, 4-aminobutyric acid,4-aminocyclohexylcarboxylic acid, succinimidyl4-(N-maleimidomethyl)cyclohexane-I-carboxy-(6-amido-caproate) (LCSMCC),succinimidyl m-maleimido-benzoylate (MBS), succinimidylN-e-maleimido-caproylate (EMCS), succinimidyl6-(beta-maleimido-propionamido) hexanoate (SMPH), succinimidylN-(a-maleimido acetate) (AMAS), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB), beta-alanine (beta-ALA),phenylglycine (PHG), 4-aminocyclohexanoic acid (ACHC),beta-(cyclopropyl) alanine (beta-CYPR), amino dodecanoic acid (ADC),alylene diols, polyethylene glycols, amino acids, and the like.

A wide variety of further linker groups are known in the art that can beuseful in the attachment of conjugate moieties to oligomeric compounds.A review of many of the useful linker groups can be found in, forexample, Antisense Research and Applications, S. T. Crooke and B.Lebleu, Eds., CRC Press, Boca Raton, Fla., 1993, p. 303-350. A disulfidelinkage has been used to link the 3′ terminus of an oligonucleotide to apeptide (Corey, et al., Science 1987, 238, 1401; Zuckermann, et al, JAm. Chem. Soc. 1988, 110, 1614; and Corey, et al., J Am. Chem. Soc.1989, 111, 8524). Nelson, et al., Nuc. Acids Res. 1989, 17, 7187describe a linking reagent for attaching biotin to the 3′-terminus of anoligonucleotide. This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol iscommercially available from Clontech Laboratories (Palo Alto, Calif.)under the name 3′-Amine. It is also commercially available under thename 3′-Amino-Modifier reagent from Glen Research Corporation (Sterling,Va.). This reagent was also utilized to link a peptide to anoligonucleotide as reported by Judy, et al., Tetrahedron Letters 1991,32, 879. A similar commercial reagent for linking to the 5′-terminus ofan oligonucleotide is 5′-Amino-Modifier C₆. These reagents are availablefrom Glen Research Corporation (Sterling, Va.). These compounds orsimilar ones were utilized by Krieg, et al, Antisense Research andDevelopment 1991, 1, 161 to link fluorescein to the 5′-terminus of anoligonucleotide. Other compounds such as acridine have been attached tothe 3′-terminal phosphate group of an oligonucleotide via apolymethylene linkage (Asseline, et al., Proc. Natl. Acad. Sci. USA1984, 81, 3297). [0074] Any of the above groups can be used as a singlelinker or in combination with one or more further linkers.

Linkers and their use in preparation of conjugates of oligomericcompounds are provided throughout the art such as in WO 96/11205 and WO98/52614 and U.S. Pat. Nos. 4,948,882; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,580,731; 5,486,603; 5,608,046; 4,587,044; 4,667,025;5,254,469; 5,245,022; 5,112,963; 5,391,723; 5,510,475; 5,512,667;5,574,142; 5,684,142; 5,770,716; 6,096,875; 6,335,432; and 6,335,437,Wo2012/083046 each of which is incorporated by reference in itsentirety.

As used herein, a physiologically labile bond is a labile bond that iscleavable under conditions normally encountered or analogous to thoseencountered within a mammalian body (also referred to as a cleavablelinker, illustrated as region B in FIGS. 12 and 13).

Physiologically labile linkage groups are selected such that theyundergo a chemical transformation (e.g., cleavage) when present incertain physiological conditions. Mammalian intracellular conditionsinclude chemical conditions such as pH, temperature, oxidative orreductive conditions or agents, and salt concentration found in oranalogous to those encountered in mammalian cells. Mammalianintracellular conditions also include the presence of enzymatic activitynormally present in a mammalian cell such as from proteolytic orhydrolytic enzymes. In some embodiments, the cleavable linker issusceptible to nuclease(s) which may for example, be expressed in thetarget cell—and as such, as detailed herein, the linker may be a shortregion (e.g. 1-10) phosphodiester linked nucleosides, such as DNAnucleosides.

Chemical transformation (cleavage of the labile bond) may be initiatedby the addition of a pharmaceutically acceptable agent to the cell ormay occur spontaneously when a molecule containing the labile bondreaches an appropriate intra- and/or extra-cellular environment. Forexample, a pH labile bond may be cleaved when the molecule enters anacidified endosome. Thus, a pH labile bond may be considered to be anendosomal cleavable bond. Enzyme cleavable bonds may be cleaved whenexposed to enzymes such as those present in an endosome or lysosome orin the cytoplasm. A disulfide bond may be cleaved when the moleculeenters the more reducing environment of the cell cytoplasm. Thus, adisulfide may be considered to be a cytoplasmic cleavable bond. As usedherein, a pH-labile bond is a labile bond that is selectively brokenunder acidic conditions (pH<7). Such bonds may also be termedendosomally labile bonds, since cell endosomes and lysosomes have a pHless than 7.

Oligomer Linked Biocleavable Conjugates

The oligomeric compound may optionally, comprise a second region (regionB) which is positioned between the oligomer (referred to as region A)and the conjugate (referred to as region C) See FIGS. 12 and 13 forillustrations). Region B may be a linker such as a cleavable linker(also referred to as a physiologically labile linkage). NucleaseSusceptible Physiological Labile Linkages: In some embodiments, theoligomer (also referred to as oligomeric compound) of the invention (orconjugate) comprises three regions:

-   -   i) a first region (region A), which comprises 10-18 contiguous        nucleotides;    -   ii) a second region (region B) which comprises a biocleavable        linker    -   iii) a third region (C) which comprises a conjugate moiety, a        targeting moiety, an activation moiety, wherein the third region        is covalent linked to the second region.

In some embodiments, region B may be a phosphate nucleotide linker. Forexample such linkers may be used when the conjugate is a lipophilicconjugate, such as a lipid, a fatty acid, sterol, such as cholesterol ortocopherol. Phosphate nucleotide linkers may also be used for otherconjugates, for example carbohydrate conjugates, such as GalNAc.

Peptide Linkers

In some embodiments, the biocleable linker (region B) is a peptide, suchas a trilysine peptide linker which may be used in a polyGalNAcconjugate, such a triGalNAc conjugate. See also the peptide biradicalsmentioned herein.

Other linkers known in the art which may be used, include disulfidelinkers.

Phosphate Nucleotide Linkers

In some embodiments, region B comprises between 1-6 nucleotides, whichis covalently linked to the 5′ or 3′ nucleotide of the first region,such as via a internucleoside linkage group such as a phosphodiesterlinkage, wherein either

-   -   a. the internucleoside linkage between the first and second        region is a phosphodiester linkage and the nucleoside of the        second region [such as immediately] adjacent to the first region        is either DNA or RNA; and/or    -   b. at least 1 nucleoside of the second region is a        phosphodiester linked DNA or RNA nucleoside;

In some embodiments, region A and region B form a single contiguousnucleotide sequence of 12-22 nucleotides in length.

In some aspects the internucleoside linkage between the first and secondregions may be considered part of the second region.

In some embodiments, there is a phosphorus containing linkage groupbetween the second and third region. The phosphorus linkage group, may,for example, be a phosphate (phosphodiester), a phosphorothioate, aphosphorodithioate or a boranophosphate group. In some embodiments, thisphosphorus containing linkage group is positioned between the secondregion and a linker region which is attached to the third region. Insome embodiments, the phosphate group is a phosphodiester.

Therefore, in some aspects the oligomeric compound comprises at leasttwo phosphodiester groups, wherein at least one is as according to theabove statement of invention, and the other is positioned between thesecond and third regions, optionally between a linker group and thesecond region.

In some embodiments, the third region is an activation group, such as anactivation group for use in conjugation. In this respect, the inventionalso provides activated oligomers comprising region A and B and aactivation group, e.g an intermediate which is suitable for subsequentlinking to the third region, such as suitable for conjugation.

In some embodiments, the third region is a reactive group, such as areactive group for use in conjugation. In this respect, the inventionalso provides oligomers comprising region A and B and a reactive group,e.g an intermediate which is suitable for subsequent linking to thethird region, such as suitable for conjugation. The reactive group may,in some embodiments comprise an amine of alcohol group, such as an aminegroup.

In some embodiments region A comprises at least one, such as 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21internucleoside linkages other than phosphodiester, such asinternucleoside linkages which are (optionally independently] selectedfrom the group consisting of phosphorothioate, phosphorodithioate, andboranophosphate, and methylphosphonate, such as phosphorothioate. Insome embodiments region A comprises at least one phosphorothioatelinkage. In some embodiments at least 50%, such as at least 75%, such asat least 90% of the internucleoside linkages, such as all theinternucleoside linkages within region A are other than phosphodiester,for example are phosphorothioate linkages. In some embodiments, all theinternucleoside linkages in region A are other than phosphodiester.

In some embodiments, the oligomeric compound comprises an antisenseoligonucleotide, such as an antisense oligonucleotide conjugate. Theantisense oligonucleotide may be or may comprise the first region, andoptionally the second region. In this respect, in some embodiments,region B may form part of a contiguous nucleobase sequence which iscomplementary to the (nucleic acid) target. In other embodiments, regionB may lack complementarity to the target.

Alternatively stated, in some embodiments, the invention provides anon-phosphodieser linked, such as a phosphorothioate linked,oligonucleotide (e.g. an antisense oligonucleotide) which has at leastone terminal (5′ and/or 3′) DNA or RNA nucleoside linked to the adjacentnucleoside of the oligonucleotide via a phosphodiester linkage, whereinthe terminal DNA or RNA nucleoside is further covalently linked to aconjugate moiety, a targeting moiety or a blocking moiety, optionallyvia a linker moiety.

In some embodiments, the oligomeric compound comprises an antisenseoligonucleotide, such as an antisense oligonucleotide conjugate. Theantisense oligonucleotide may be or may comprise the first region, andoptionally the second region. In this respect, in some embodiments,region B may form part of a contiguous nucleobase sequence which iscomplementary to the (nucleic acid) target. In other embodiments, regionB may lack complementarity to the target.

In some embodiments, at least two consecutive nucleosides of the secondregion are DNA nucleosides (such as at least 3 or 4 or 5 consecutive DNAnucleotides).

In such an embodiment, the oligonucleotide of the invention may bedescribed according to the following formula:5′-A-PO—B[Y)X-3′ or 3′-A-PO—B[Y)X-5′

wherein A is region A, PO is a phosphodiester linkage, B is region B, Yis an optional linkage group, and X is a conjugate, a targeting, ablocking group or a reactive or activation group.

In some embodiments, region B comprises 3′-5′ or 5′-3′: i) aphosphodiester linkage to the 5′ or 3′ nucleoside of region A, ii) a DNAor RNA nucleoside, such as a DNA nucleoside, and iii) a furtherphosphodiester linkage5′-A-PO—B—PO-3′ or 3′-A-PO—B—PO-5′

The further phosphodiester linkage link the region B nucleoside with oneor more further nucleoside, such as one or more DNA or RNA nucleosides,or may link to X (is a conjugate, a targeting or a blocking group or areactive or activation group) optionally via a linkage group (Y).

In some embodiments, region B comprises 3′-5′ or 5′-3′: i) aphosphodiester linkage to the 5′ or 3′ nucleoside of region A, ii)between 2-10 DNA or RNA phosphodiester linked nucleosides, such as a DNAnucleoside, and optionally iii) a further phosphodiester linkage:5′-A-[PO—B]n-[Y]—X3′ or 3′-A-[PO—B]n-[Y]—X5′5′-A-[PO—B]n-PO—[Y]—X3′ or 3′-A-[PO—B]n-PO—[Y]—X5′

Wherein A represent region A, [PO—B]n represents region B, wherein n is1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, PO is an optionalphosphodiester linkage group between region B and X (or Y if present).

In some embodiments the invention provides compounds according to (orcomprising) one of the following formula:5′[Region A]-PO-[region B]3′-Y—X5′[Region A]-PO-[region B]—PO 3′-Y—X5′[Region A]-PO-[region B]3′-X5′[Region A]-PO-[region B]—PO 3′-X3′[Region A]-PO-[region B]5′-Y—X3′[Region A]-PO-[region B]—PO 5′-Y—X3′[Region A]-PO-[region B]5′-X3′[Region A]-PO-[region B]—PO 5′-X

Region B, may for example comprise or consist of:5′DNA3′3′DNA5′5′DNA-PO-DNA-3′3′DNA-PO-DNA-5′5′DNA-PO-DNA-PO-DNA3′3′DNA-PO-DNA-PO-DNA5′5′DNA-PO-DNA-PO-DNA-PO-DNA3′3′DNA-PO-DNA-PO-DNA-PO-DNA5′5′DNA-PO-DNA-PO-DNA-PO-DNA-PO-DNA3′3′DNA-PO-DNA-PO-DNA-PO-DNA-PO-DNA5′

It should be recognized that phosphate linked biocleavable linkers mayemploy nucleosides other than DNA and RNA. Biocleavable nucleotidelinkers can be identified using the assays in example 6.

In some embodiments, the compound of the invention comprises abiocleavable linker (also referred to as the physiologically labilelinker, Nuclease Susceptible Physiological Labile Linkages, or nucleasesusceptible linker), for example the phosphate nucleotide linker (suchas region B) or a peptide linker, which joins the oligomer (orcontiguous nucleotide sequence or region A), to a conjugate moiety (orregion C).

The susceptibility to cleavage in the assays shown in Example 6 can beused to determine whether a linker is biocleavable or physiologicallylabile.

Biocleavable linkers according to the present invention refers tolinkers which are susceptible to cleavage in a target tissue (i.e.physiologically labile), for example liver and/or kidney. It ispreferred that the cleavage rate seen in the target tissue is greaterthan that found in blood serum. Suitable methods for determining thelevel (%) of cleavage in tissue (e.g. liver or kidney) and in serum arefound in example 6. In some embodiments, the biocleavable linker (alsoreferred to as the physiologically labile linker, or nucleasesusceptible linker), such as region B, in a compound of the invention,are at least about 20% cleaved, such as at least about 30% cleaved, suchas at least about 40% cleaved, such as at least about 50% cleaved, suchas at least about 60% cleaved, such as at least about 70% cleaved, suchas at least about 75% cleaved, in the liver or kidney homogenate assayof Example 6. In some embodiments, the cleavage (%) in serum, as used inthe assay in Example 6, is less than about 30%, is less than about 20%,such as less than about 10%, such as less than 5%, such as less thanabout 1%.

In some embodiments, which may be the same of different, thebiocleavable linker (also referred to as the physiologically labilelinker, or nuclease susceptible linker), such as region B, in a compoundof the invention, are susceptible to S1 nuclease cleavage.Susceptibility to S1 cleavage may be evaluated using the S1 nucleaseassay shown in Example 6. In some embodiments, the biocleavable linker(also referred to as the physiologically labile linker, or nucleasesusceptible linker), such as region B, in a compound of the invention,are at least about 30% cleaved, such as at least about 40% cleaved, suchas at least about 50% cleaved, such as at least about 60% cleaved, suchas at least about 70% cleaved, such as at least about 80% cleaved, suchas at least about 90% cleaved, such as at least 95% cleaved after 120min incubation with S1 nuclease according to the assay used in Example6.

Sequence Selection in the Second Region:

In some embodiments, region B does not form a complementary sequencewhen the oligonucleotide region A and B is aligned to the complementarytarget sequence.

In some embodiments, region B does form a complementary sequence whenthe oligonucleotide region A and B is aligned to the complementarytarget sequence. In this respect region A and B together may form asingle contiguous sequence which is complementary to the targetsequence.

In some embodiments, the sequence of bases in region B is selected toprovide an optimal endonuclease cleavage site, based upon thepredominant endonuclease cleavage enzymes present in the target tissueor cell or sub-cellular compartment. In this respect, by isolating cellextracts from target tissues and non-target tissues, endonucleasecleavage sequences for use in region B may be selected based upon apreferential cleavage activity in the desired target cell (e.g.liver/hepatocytes) as compared to a non-target cell (e.g. kidney). Inthis respect, the potency of the compound for target down-regulation maybe optimized for the desired tissue/cell.

In some embodiments region B comprises a dinucleotide of sequence AA,AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG, wherein Cmay be 5-methylcytosine, and/or T may be replaced with U. In someembodiments region B comprises a trinucleotide of sequence AAA, AAT,AAC, AAG, ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG,TAA, TAT, TAC, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT,TGC, TGG, CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC, CCG,CGA, CGT, CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT,GCC, GCG, GGA, GGT, GGC, and GGG wherein C may be 5-methylcytosineand/or T may be replaced with U. In some embodiments region B comprisesa trinucleotide of sequence AAAX, AATX, AACX, AAGX, ATAX, ATTX, ATCX,ATGX, ACAX, ACTX, ACCX, ACGX, AGAX, AGTX, AGCX, AGGX, TAAX, TATX, TACX,TAGX, TTAX, TTTX, TTCX, TAGX, TCAX, TCTX, TCCX, TCGX, TGAX, TGTX, TGCX,TGGX, CAAX, CATX, CACX, CAGX, CTAX, CTGX, CTCX, CTTX, CCAX, CCTX, CCCX,CCGX, CGAX, CGTX, CGCX, CGGX, GAAX, GATX, GACX, CAGX, GTAX, GTTX, GTCX,GTGX, GCAX, GCTX, GCCX, GCGX, GGAX, GGTX, GGCX, and GGGX, wherein X maybe selected from the group consisting of A, T, U, G, C and analoguesthereof, wherein C may be 5-methylcytosine and/or T may be replaced withU. It will be recognized that when referring to (naturally occurring)nucleobases A, T, U, G, C, these may be substituted with nucleobaseanalogues which function as the equivalent natural nucleobase (e.g. basepair with the complementary nucleoside). In some embodiments region Bdoes not comprise a T or U.

Amino Alkyl Intermediates

The invention further provides for the LNA oligomer intermediates whichcomprise an antisense LNA oligomer which comprises an (e.g. terminal, 5′or 3′) amino alkyl linker, such as a C2-C36 amino alkyl group, forexample a C6 to C12 amino alkyl group, including for example C6 and C12amino alkyl groups. The amino alkyl group may be added to the LNAoligomer as part of standard oligonucleotide synthesis, for exampleusing a (e.g. protected) amino alkyl phosphoramidite. The linkage groupbetween the amino alkyl and the LNA oligomer may for example be aphosphorothioate or a phosphodiester, or one of the other nucleosidelinkage groups referred to herein, for example. The amino alkyl groupmay be covalently linked to, for example, the 5′ or 3′ of the LNAoligomer, such as by the nucleoside linkage group, such asphosphorothioate or phosphodiester linkage.

The invention also provides a method of synthesis of the LNA oligomercomprising the sequential synthesis of the LNA oligomer, such as solidphase oligonucleotide synthesis, comprising the step of adding a aminoalkyl group to the oligomer, such as e.g. during the first or last roundof oligonucleotide synthesis. The method of synthesis may furthercomprise the step of reacting the a conjugate to the amino alkyl-LNAoligomer (the conjugation step). The a conjugate may comprise suitablelinkers and/or branch point groups, and optionally further conjugategroups, such as hydrophobic or lipophilic groups, as described herein.The conjugation step may be performed whilst the oligomer is bound tothe solid support (e.g. after oligonucleotide synthesis, but prior toelution of the oligomer from the solid support), or subsequently (i.e.after elution). The invention provides for the use of an amino alkyllinker in the synthesis of the oligomer of the invention.

Method of Manufacture/Synthesis

The invention provides for a method of synthesizing (or manufacture) ofan oligomeric compound, such as the oligomeric compound of theinvention, said method comprising either:

-   -   a) a step of providing a [solid phase] oligonucleotide synthesis        support to which one of the following is attached [third        region]:        -   i) a linker group (—Y—)        -   ii) a group selected from the group consisting of a            conjugate, a targeting group, a blocking group, a reactive            group [e.g. an amine or an alcohol] or an activation group            (X—)        -   iii) an —Y—X group    -   and    -   b) a step of [sequential] oligonucleotide synthesis of region B        followed by region A, and/I or:    -   c) a step of [sequential] oligonucleotide synthesis of a first        region (A) and a second region (B), wherein the synthesis step        is followed by    -   d) a step of adding a third region [phosphoramidite comprising]        -   i) a linker group (—Y—)        -   ii) a group selected from the group consisting of a            conjugate, a targeting group, a blocking group, a reactive            group [e.g. an amine or an alcohol] or an activation group            (X—)        -   iii) an —Y—X group followed by    -   e) the cleavage of the oligomeric compound from the [solid        phase] support wherein, optionally said method further comprises        a further step selected from:    -   f) wherein the third group is an activation group, the step of        activating the activation group to produce a reactive group,        followed by adding a conjugate, a blocking, or targeting group        to the reactive group, optionally via a linker group (Y);    -   g) wherein the third region is a reactive group, the step of        adding a conjugate, a blocking, or targeting group to the        reactive group, optionally via a linker group (Y).    -   h) wherein the third region is a linker group (Y), the step of        adding a conjugate, a blocking, or targeting group to the linker        group (Y)    -   wherein steps f), g) or h) are performed either prior to or        subsequent to cleavage of the oligomeric compound from the        oligonucleotide synthesis support. In some embodiments, the        method may be performed using standard phosphoramidite        chemistry, and as such the region X and/or region X or region X        and Y may be provided, prior to incorporation into the oligomer,        as a phosphoramidite. Please see FIGS. 5-10 which illustrate        non-limiting aspects of the method of the invention.

The invention provides for a method of synthesizing (or manufacture) ofan oligomeric compound, such as the oligomeric compound of theinvention, said method comprising

a step of sequential oligonucleotide synthesis of a first region (A) anda second region (B), wherein the synthesis step is followed by a step ofadding a third region phosphoramidite comprising region X (also referredto as region C) or Y, such as a region comprising a group selected fromthe group consisting of a conjugate, a targeting group, a blockinggroup, a functional group, a reactive group (e.g. an amine or analcohol) or an activation group (X), or an —Y—X group followed by thecleavage of the oligomeric compound from the [solid phase] support.

It is however recognized that the region X or X—Y may be added after thecleavage from the solid support. Alternatively, the method of synthesismay comprise the steps of synthesizing a first (A), and optionallysecond region (B), followed by the cleavage of the oligomer from thesupport, with a subsequent step of adding a third region, such as X orX—Y group to the oligomer. The addition of the third region may beachieved, by example, by adding an amino phosphoramidite unit in thefinal step of oligomer synthesis (on the support), which can, aftercleavage from the support, be used to join to the X or X—Y group,optionally via an activation group on the X or Y (when present) group.In the embodiments where the cleavable linker is not a nucleotideregion, region B may be a non-nucleotide cleavable linker for example apeptide linker, which may form part of region X (also referred to asregion C) or be region Y (or part thereof).

In some embodiments of the method, region X (such as C) or (X-Y), suchas the conjugate (e.g. a GalNAc conjugate) comprises an activationgroup, (an activated functional group) and in the method of synthesisthe activated conjugate (or region x, or X-Y) is added to the first andsecond regions, such as an amino linked oligomer. The amino group may beadded to the oligomer by standard phosphoramidite chemistry, for exampleas the final step of oligomer synthesis (which typically will result inamino group at the 5′ end of the oligomer). For example during the laststep of the oligonucleotide synthesis a protected amino-alkylphosphoramidite is used, for example a TFA-aminoC6 phosphoramidite(6-(Trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite).

Region X (or region C as referred to herein), such as the conjugate(e.g. a GalNAc conjugate) may be activated via NHS ester method and thenthe aminolinked oligomer is added. For example a N-hydroxysuccinimide(NHS) may be used as activating group for region X (or region C, such asa conjugate, such as a GalNAc conjugate moiety.

The invention provides an oligomer prepared by the method of theinvention.

In some embodiments, region X and/or region X or region X and Y may becovalently joined (linked) to region B via a phosphate nucleosidelinkage, such as those described herein, including phosphodiester orphosphorothioate, or via an alternative group, such as a triazol group.

In some embodiments, the internucleoside linkage between the first andsecond region is a phosphodiester linked to the first (or only) DNA orRNA nucleoside of the second region, or region B comprises at least onephosphodiester linked DNA or RNA nucleoside.

The second region may, in some embodiments, comprise further DNA or RNAnucleosides which may be phosphodester linked. The second region isfurther covalently linked to a third region which may, for example, be aconjugate, a targeting group a reactive group, and/or a blocking group.

In some aspects, the present invention is based upon the provision of alabile region, the second region, linking the first region, e.g. anantisense oligonucleotide, and a conjugate or functional group, e.g. atargeting or blocking group. The labile region comprises at least onephosphodiester linked nucleoside, such as a DNA or RNA nucleoside, suchas 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphodiester linked nucleosides,such as DNA or RNA. In some embodiments, the oligomeric compoundcomprises a cleavable (labile) linker. In this respect the cleavablelinker is preferably present in region B (or in some embodiments,between region A and B).

Alternatively stated, in some embodiments, the invention provides anon-phosphodiester linked, such as a phosphorothioate linked,oligonucleotide (e.g. an antisense oligonucleotide) which has at leastone terminal (5′ and/or 3′) DNA or RNA nucleoside linked to the adjacentnucleoside of the oligonucleotide via a phosphodiester linkage, whereinthe terminal DNA or RNA nucleoside is further covalently linked to aconjugate moiety, a targeting moiety or a blocking moiety, optionallyvia a linker moiety.

Compositions

The oligomer or oligomer conjugates of the invention may be used inpharmaceutical formulations and compositions. Suitably, suchcompositions comprise a pharmaceutically acceptable diluent, carrier,salt or adjuvant. WO2007/031091 provides suitable and preferredpharmaceutically acceptable diluent, carrier and adjuvants—which arehereby incorporated by reference. Suitable dosages, formulations,administration routes, compositions, dosage forms, combinations withother therapeutic agents, pro-drug formulations are also provided inWO2007/031091—which are also hereby incorporated by reference.

Applications

The oligomers or oligomer conjugates of the invention may be utilized asresearch reagents for, for example, diagnostics, therapeutics andprophylaxis.

In research, such oligomers may be used to specifically inhibit thesynthesis of PCSK9 protein (typically by degrading or inhibiting themRNA and thereby prevent protein formation) in cells and experimentalanimals thereby facilitating functional analysis of the target or anappraisal of its usefulness as a target for therapeutic intervention.

In diagnostics the oligomers may be used to detect and quantitate PCSK9expression in cell and tissues by northern blotting, in-situhybridisation or similar techniques.

For therapeutics, an animal or a human, suspected of having a disease ordisorder, which can be treated by modulating the expression of PCSK9 istreated by administering oligomeric compounds in accordance with thisinvention. Further provided are methods of treating a mammal, such astreating a human, suspected of having or being prone to a disease orcondition, associated with expression of PCSK9 by administering atherapeutically or prophylactically effective amount of one or more ofthe oligomers or compositions of the invention. The oligomer, aconjugate or a pharmaceutical composition according to the invention istypically administered in an effective amount.

The invention also provides for the use of the compound or conjugate ofthe invention as described for the manufacture of a medicament for thetreatment of a disorder as referred to herein, or for a method of thetreatment of as a disorder as referred to herein.

The invention also provides for a method for treating a disorder asreferred to herein said method comprising administering a compoundaccording to the invention as herein described, and/or a conjugateaccording to the invention, and/or a pharmaceutical compositionaccording to the invention to a patient in need thereof.

Medical Indications

The oligomers, oligomer conjugates and other compositions according tothe invention can be used for the treatment of conditions associatedwith over expression or expression of mutated version of the PCSK9.

The invention further provides use of a compound of the invention in themanufacture of a medicament for the treatment of a disease, disorder orcondition as referred to herein.

Generally stated, one aspect of the invention is directed to a method oftreating a mammal suffering from or susceptible to conditions associatedwith abnormal levels and/or activity of PCSK9, comprising administeringto the mammal and therapeutically effective amount of an oligomer oroligomer conjugate targeted to PCSK9 that comprises one or more LNAunits. The oligomer, a conjugate or a pharmaceutical compositionaccording to the invention is typically administered in an effectiveamount.

The disease or disorder, as referred to herein, may, in some embodimentsbe associated with a mutation in the PCSK9 gene or a gene whose proteinproduct is associated with or interacts with PCSK9. Therefore, in someembodiments, the target mRNA is a mutated form of the PCSK9 sequence.

An interesting aspect of the invention is directed to the use of anoligomer (compound) as defined herein or a conjugate as defined hereinfor the preparation of a medicament for the treatment of a disease,disorder or condition as referred to herein.

The methods of the invention are preferably employed for treatment orprophylaxis against diseases caused by abnormal levels and/or activityof PCSK9.

Alternatively stated, In some embodiments, the invention is furthermoredirected to a method for treating abnormal levels and/or activity ofPCSK9, said method comprising administering a oligomer of the invention,or a conjugate of the invention or a pharmaceutical composition of theinvention to a patient in need thereof.

The invention also relates to an oligomer, a composition or a conjugateas defined herein for use as a medicament.

The invention further relates to use of a compound, composition, or aconjugate as defined herein for the manufacture of a medicament for thetreatment of abnormal levels and/or activity of PCSK9 or expression ofmutant forms of PCSK9 (such as allelic variants, such as thoseassociated with one of the diseases referred to herein).

Moreover, the invention relates to a method of treating a subjectsuffering from a disease or condition such as those referred to herein.

A patient who is in need of treatment is a patient suffering from orlikely to suffer from the disease or disorder.

In some embodiments, the term ‘treatment’ as used herein refers to bothtreatment of an existing disease (e.g. a disease or disorder as hereinreferred to), or prevention of a disease, i.e. prophylaxis. It willtherefore be recognised that treatment as referred to herein may, Insome embodiments, be prophylactic.

In one embodiment, the invention relates to compounds or compositionscomprising compounds for treatment of hypercholesterolemia and relateddisorders, or methods of treatment using such compounds or compositionsfor treating hypercholesterolemia and related disorders, wherein theterm “related disorders” when referring to hypercholesterolemia refersto one or more of the conditions selected from the group consisting of:atherosclerosis, hyperlipidemia, hypercholesterolemia, familiarhypercholesterolemia e.g. gain of function mutations in PCSK9, HDL/LDLcholesterol imbalance, dyslipidemias, e.g., familial hyperlipidemia(FCHL) or familial hypercholesterolemia (FHC), acquired hyperlipidemia,statin-resistant hypercholesterolemia, coronary artery disease (CAD),and coronary heart disease (CHD).

Combination Treatments

In some embodiments the compound of the invention is for use in acombination treatment with another therapeutic agent. E.g. inhibitors ofHMG CoA reductase, such as statins for example are widely used in thetreatment of metabolic disease (see WO2009/043354, hereby incorporatedby reference for examples of combination treatments). Combinationtreatments may be other cholesterol lowering compounds, such as acompound selected from the group consisting of bile salt sequesteringresins (e.g., cholestyramine, colestipol, and colesevelamhydrochloride), HMGCoA-reductase inhibitors (e.g., lovastatin,cerivastatin, pravastatin, atorvastatin, simvastatin, rosuvastatin, andfluvastatin), nicotinic acid, fibric acid derivatives (e.g., clofibrate,gemfibrozil, fenofibrate, bezafibrate, and ciprofibrate), probucol,neomycin, dextrothyroxine, plant-stanol esters, cholesterol absorptioninhibitors (e.g., ezetimibe), implitapide, inhibitors of bile acidtransporters (apical sodium-dependent bile acid transporters),regulators of hepatic CYP7a, estrogen replacement therapeutics (e.g.,tamoxifen), and anti-inflammatories (e.g., glucocorticoids).Combinations with statins may be particularly preferred.

Specific Embodiments of the Invention

-   1. An antisense oligonucleotide conjugate comprising    -   a. an antisense oligomer (A) of between 12-22 nucleotides in        length, which comprises a contiguous sequence of 10-16        nucleotides which are complementary to a corresponding length of        SEQ ID NO 30 or 31 or 32 or 33 or 34 or 45, and    -   b. at least one non-nucleotide or non-polynucleotide conjugate        moiety (C) covalently attached to said oligomer (A).-   2. The oligonucleotide conjugate according to embodiment 1, wherein    the antisense oligomer comprises a contiguous sequence selected from    the group consisting of SEQ ID NO 25, 26, 27, 28, 29 and 44.-   3. The oligonucleotide conjugate according any one of embodiments 1    or 2, wherein the antisense oligomer targets PCSK9.-   4. The oligonucleotide conjugate according to any one of 1 to 3,    wherein the antisense oligomer comprises affinity enhancing    nucleotide analogues.-   5. The oligonucleotide conjugate according to embodiment 4, wherein    the nucleotide analogues are sugar modified nucleotides, such as    sugar modified nucleotides independently or dependently selected    from the group consisting of: Locked Nucleic Acid (LNA) units;    2′-O-alkyl-RNA units, 2′-OMe-RNA units, 2′-amino-DNA units, and    2′-fluoro-DNA units.-   6. The oligonucleotide conjugate according to embodiment 4 or 5,    wherein the nucleotide analogues comprise or are Locked Nucleic Acid    (LNA) units.-   7. The oligonucleotide conjugate according to any one of embodiments    1 to 6, wherein the antisense oligomer is a gapmer.-   8. The oligonucleotide conjugate according to embodiment 7, wherein    the gapmer comprise a wing on each side (5′ and 3′) of 2 to 4    nucleotide analogues, preferably LNA analogues.-   9. The oligonucleotide conjugate according to embodiment 7 or 8,    wherein the gapmer design is selected from the group consisting of    2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-2, 2-8-4, 2-9-2, 2-9-3, 3-9-2,    3-9-3, 3-9-4, 4-9-3, 2-10-2, 2-10-3, 3-10-2, 3-10-3, 3-10-4, 4-10-3,    2-11-2, 2-11-3, 3-11-2, 3-11-3, 3-11-4, 4-11-3 and 4-11-4.-   10. The oligonucleotide conjugate according to any one of the    embodiments 7 to 9, wherein the gapmer design is selected from the    group consisting of 2-8-3, 3-8-3, 3-9-4, 3-10-3, 2-11-2, 2-11-3 and    3-11-2.-   11. The oligonucleotide conjugate according to any one of the    embodiments 1 to 10, wherein the oligomer comprises a contiguous    sequence of 13, 14, 15 or 16 nucleotides.-   12. The oligonucleotide conjugate according to any one of the    embodiments 1 to 11, wherein the oligomer comprises one or more    nucleoside linkages selected from the group consisting of    phosphorothioate, phosphorodithioate and boranophosphate.-   13. The oligonucleotide conjugate according to any one of    embodiments 1 to 12, wherein the oligomer comprises or consist of    phosphorothioate nucleoside linkages.-   14. The oligonucleotide conjugate according to any one of the    embodiments 1 to 12, wherein the oligomer comprises a contiguous    sequence selected from the group consisting of SEQ ID NO 1, 2, 3, 4,    5, 6, 7, and 8.-   15. The oligonucleotide conjugate according to any one of the    embodiments 1 to 14, wherein the conjugate moiety (C) is selected    from the group consisting of or a carbohydrate, such as GalNAc or a    GalNAc cluster; a lipophilic group, such as a lipid, a fatty acid; a    sterol, such as cholesterol or tocopherol; or a statin.-   16. The antisense oligonucleotide conjugate according to any one of    embodiments 1 to 15, wherein the conjugate moiety (C) enhances    delivery and/or uptake to liver cells.-   17. The antisense oligonucleotide conjugate according to any one of    embodiments 1 to 16, wherein the conjugate moiety (C) comprises a    sterol such as tocophorol, cholesterol, such as those shown as Conj    5, Conj 5, Conj 6 or Conj 6a.-   18. The antisense oligonucleotide conjugate according to embodiment    1 to 16, wherein the conjugate moiety (C) comprises a carbohydrate    such as GalNAc or trivalent GalNAc, such as those shown as Conj 1,    2, 3 or 4, or 1a, 2a, 3a or 4a.-   19. The antisense oligonucleotide conjugate according to embodiment    18, wherein the conjugate moiety (C) comprises Conj 2a.-   20. The antisense oligonucleotide conjugate according to any one of    embodiments 1 to 19, which is selected from the group consisting of    SEQ ID NO 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,    and 24.-   21. The antisense oligonucleotide conjugate according to any one of    embodiments 1 to 20, wherein the antisense oligomer (A) is    conjugated to the conjugate moiety (C) via a linker region    positioned between the contiguous sequence of the oligomer and the    conjugate moiety (B and/or Y).-   22. The antisense oligonucleotide conjugate according to embodiment    21, wherein the linker is selected from the group consisting of    amino alkyl linkers, phosphate nucleotide linkers and peptide    linkers.-   23. The antisense oligonucleotide conjugate according to embodiment    21 or 22, wherein the linker is selected from a C6 to C12 amino    alkyl groups.-   24. The antisense oligonucleotide conjugate according to embodiment    21 or 22, wherein the linker is a bioclevable phosphate nucleotide    linker comprising between 1 to 6 nucleotides.-   25. The antisense oligonucleotide conjugate according to any of    embodiments 21 to 24, wherein the linker (B) is a phosphodiester    nucleotide linkage comprising one or more contiguous DNA    phosphodiester nucleotides, such as 1, 2, 3, 4, 5, or 6 DNA    phosphodiester nucleotides which are contiguous with the 5′ or 3′    end of the contiguous sequence of the oligomer, and which may or may    not form complementary base pairing with the PCSK9 target sequence.-   26. The antisense oligonucleotide conjugate according to embodiment    24 or 25, wherein the phosphodiester nucleotide linkage (or    biocleavable linker) comprises 1, 2 or 3 DNA phosphodiester    nucleotides, such as two DNA phosphodiester nucleotides, such as a    5′ CA 3′ dinucleotide.-   27. A oligomer of between 12-22 nucleotides in length, which either    comprises    -   a. a contiguous sequence of 16 nucleotides which are        complementary to a corresponding length of SEQ ID NO 31, or    -   b. a contiguous sequence of 10-16 nucleotides which are        complementary to a corresponding length of SEQ ID NO 33 or 34 or        45.-   28. The oligomer according to embodiment 27, which comprises a    contiguous sequence selected from the group consisting of SEQ ID NO    26, 27, 28, 29 and 44.-   29. The oligomer according any one of embodiments 27 or 28, wherein    the oligomer targets PCSK9.-   30. The oligomer according to any one of embodiments 27 to 29    wherein the contiguous sequence comprises affinity enhancing    nucleotide analogues.-   31. The oligomer according to embodiment 30, wherein the nucleotide    analogues are sugar modified nucleotides, such as sugar modified    nucleotides independently or dependently selected from the group    consisting of: Locked Nucleic Acid (LNA) units; 2′-O-alkyl-RNA    units, 2′-OMe-RNA units, 2′-amino-DNA units, and 2′-fluoro-DNA    units.-   32. The oligomer according to embodiment 30 or 31, wherein the    nucleotide analogues comprise or are Locked Nucleic Acid (LNA)    units.-   33. The oligomer according to any one of embodiments 27 to 32, which    is a gapmer, such as a Locked Nucleic Acid gapmer oligonucleotide.-   34. The oligonucleotide conjugate according to embodiment 33,    wherein the gapmer comprise a wing on each side (5′ and 3′) of 2 to    4 nucleotide analogues, preferably LNA analogues.-   35. The oligonucleotide conjugate according to embodiment 33 or 34,    wherein the gapmer design is selected from the group consisting of    2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-2, 2-8-4, 2-9-2, 2-9-3, 3-9-2,    3-9-3, 3-9-4, 4-9-3, 2-10-2, 2-10-3, 3-10-2, 3-10-3, 3-10-4, 4-10-3,    2-11-2, 2-11-3, 3-11-2, 3-11-3, 3-11-4, 4-11-3 and 4-11-4.-   36. The oligonucleotide conjugate according to any one of the    embodiments 33 to 35, wherein the gapmer design is selected from the    group consisting of 2-8-3, 3-8-3, 3-9-4, 3-10-3, 2-11-2, 2-11-3 and    3-11-2.-   37. The oligomer according to any one of embodiments 27 to 36,    wherein the oligomer comprises a contiguous sequence of 13, 14, 15    or 16 nucleotides.-   38. The oligomer according to any one of embodiments 27 to 37,    wherein the oligomer comprises one or more nucleoside linkages    selected from the group consisting of phosphorothioate,    phosphorodithioate and boranophosphate.-   39. The oligomer according to any one of embodiments 27 to 38,    wherein the oligomer comprises or consist of phosphorothioate    nucleoside linkages.-   40. Then oligomer according to any one of embodiments 27 to 38,    which comprises a contiguous sequence selected from the group    consisting of SEQ ID NO 2, 3, 4, 5, 6, 7, and 8.-   41. A pharmaceutical composition comprising the oligomer or    antisense oligonucleotide conjugate according to any one of    embodiments 1 to 40 and a pharmaceutically acceptable diluent,    carrier, salt or adjuvant.-   42. The oligomer or antisense oligonucleotide conjugate or    pharmaceutical composition according to any one of embodiments 1 to    41, for use as a medicament, such as for the treatment of    hypercholesterolemia or related disorder, such as a disorder    selected from the group consisting of atherosclerosis,    hyperlipidemia, hypercholesterolemia, familiar hypercholesterolemia    e.g. gain of function mutations in PCSK9, HDL/LDL cholesterol    imbalance, dyslipidemias, e.g., familial hyperlipidemia (FCHL) or    familial hypercholesterolemia (FHC), acquired hyperlipidemia,    statin-resistant hypercholesterolemia, coronary artery disease    (CAD), and coronary heart disease (CHD).-   43. The use of an oligomer or antisense oligonucleotide conjugate or    pharmaceutical composition according to any one of the embodiments 1    to 41, for the manufacture of a medicament for the treatment of    hypercholesterolemia or a related disorder, such as a disorder    selected from the group consisting of atherosclerosis,    hyperlipidemia, hypercholesterolemia, familiar hypercholesterolemia    e.g. gain of function mutations in PCSK9, HDL/LDL cholesterol    imbalance, dyslipidemias, e.g., familial hyperlipidemia (FCHL) or    familial hypercholesterolemia (FHC), acquired hyperlipidemia,    statin-resistant hypercholesterolemia, coronary artery disease    (CAD), and coronary heart disease (CHD).-   44. A method of treating hypercholesterolemia or a related disorder,    such as a disorder selected from the group consisting    atherosclerosis, hyperlipidemia, hypercholesterolemia, familiar    hypercholesterolemia e.g. gain of function mutations in PCSK9,    HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familial    hypercholesterolemia (FHC), acquired hyperlipidemia,    statin-resistant hypercholesterolemia, coronary artery disease    (CAD), and coronary heart disease (CHD), said method comprising    administering an effective amount of an oligomer or antisense    oligonucleotide conjugate or pharmaceutical composition according to    any one of the embodiments 1 to 41, to a patient suffering from, or    likely to suffer from hypercholesterolemia or a related disorder.-   45. A in vivo or in vitro method for the inhibition of PCSK9 in a    cell which is expressing PCSK9, said method comprising administering    an oligomer or antisense oligonucleotide conjugate or pharmaceutical    composition according to any one of the embodiments 1 to 41, to said    cell so as to inhibit PCSK9 in said cell.

EXAMPLES

Oligonucleotides were synthesized on uridine universal supports usingthe phosphoramidite approach on an Expedite 8900/MOSS synthesizer(Multiple Oligonucleotide Synthesis System) or Oligomaker 48 at 4 or 1μmol scale, respectively. At the end of the synthesis, theoligonucleotides were cleaved from the solid support using aqueousammonia for 5-16 hours at 60° C. The oligonucleotides were purified byreverse phase HPLC (RP-HPLC) or by solid phase extractions andcharacterized by UPLC, and the molecular mass was further confirmed byESI-MS. See below for more details.

Elongation of the Oligonucleotide

The coupling of β-cyanoethyl-phosphoramidites (DNA-A(Bz), DNA-G(ibu),DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz), LNA-A(Bz), LNA-G(dmf), LNA-T orC6-S—S—C6 linker) is performed by using a solution of 0.1 M of the5′-O-DMT-protected amidite in acetonitrile and DCI(4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator. For thefinal cycle a commercially available C6-linked cholesterolphosphoramidite was used at 0.1 M in DCM. Thiolation for introduction ofphosphorthioate linkages is carried out by using xanthane hydride (0.01M in acetonitrile/pyridine 9:1). Phosphordiester linkages are introducedusing 0.02 M iodine in THF/Pyridine/water 7:2:1. The rest of thereagents are the ones typically used for oligonucleotide synthesis. Forpost solid phase synthesis conjugation a commercially available C6aminolinker phorphoramidite was used in the last cycle of the solidphase synthesis and after deprotection and cleavage from the solidsupport the aminolinked deprotected oligonucleotide was isolated. Theconjugates was introduced via activation of the functional group usingstandard synthesis methods.

Purification by RP-HPLC:

The crude compounds were purified by preparative RP-HPLC on a PhenomenexJupiter C18 10μ 150×10 mm column. 0.1 M ammonium acetate pH 8 andacetonitrile was used as buffers at a flow rate of 5 mL/min. Thecollected fractions were lyophilized to give the purified compoundtypically as a white solid.

Abbreviations

DCI: 4,5-Dicyanoimidazole

DCM: Dichloromethane

DMF: Dimethylformamide

DMT: 4,4′-Dimethoxytrityl

THF: Tetrahydrofurane

Bz: Benzoyl

Ibu: Isobutyryl

RP-HPLC: Reverse phase high performance liquid chromatography

The compounds synthesized are shown in Table 1 and are also illustratedin the Figures.

Example 1 New PCSK9 Target Motif Discovery

521 anti-PCSK9 antisense oligonucleotides—all with three locked nucleicacids flanking ten DNAs, i.e with 16-mer LNA gap-mer design—specific forhuman and primate PCSK9 were designed and synthesized. The human cellline 15PC3 was incubated for three days with either mock or the lockednucleic acid-modified oligonucleotides targeted to human PCSK9 atconcentration 0.3 μM. Each anti-PCSK9 oligonucleotide was tested inthree independent experiments. PCSK9 mRNA levels were quantitated fromextracted RNA using real-time PCR as described, and presented normalizedto β-actin mRNA and relative to average levels in twelve mock treatedsamples in FIG. 8, with a close-up of a sub-set of the most potentmolecules in FIG. 9.

Example 2 In Vitro mRNA Knockdown

The human cell line 15PC3 was incubated for 3 days with either mock orlocked nucleic acid modified oligonucleotides with SEQ IDs 1 to 8targeted to human PCSK9 at concentrations 0.0012, 0.06, 0.3 and 1.5 μM.PCSK9 mRNA levels were quantitated from extracted RNA using real-timePCR as described, and presented relative to average levels in four mocktreated samples in FIG. 10. For each oligonucleotide, potency,quantified as half maximal effective concentration (EC50), wasdetermined by least squares fitting of the Hill equation intwo-parameter logistic form with lower limit fixed at 0% and upper limitfixed at 100%, as EC50=estimate±standard deviation.

Example 3—In Vivo ALT Levels

Four week old female NMRI mice (Taconic, Denmark), weighingapproximately 20 g at arrival, were injected intravenously once witheither saline or locked nucleic acid-modified, cholesterol-conjugated,oligonucleotides with SEQ IDs 9 to 16 targeted to human PCSK9 at doses7.5 and 15 mg/kg. The mice were sacrificed 7 days followingadministration and serum levels of alanine aminotransferase (ALT)determined using an enzymatic assay (Horiba ABX Diagnostics). For eachtreatment group of five mice, mean and standard deviations werecalculated and presented in FIG. 11 relative to mean levels in salinetreated mice. ALT rises were noted at both concentrations for some, butnot all, cholesterol conjugated molecules. Several of the compounds,such as SEQ ID NO 9 and 10, did not enhance ALT in mice in a clinicallymeaningful manner even when cholesterol was used as a conjugate toenhance the uptake of compounds in the liver.

Example 4: Non-Human Primate Study

The primary objective for this study was to investigate selected lipidmarkers over 7 weeks after a single slow bolus injection of anti-PCSK9LNA compounds to cynomolgus monkeys and assess the potential toxicity ofcompounds in monkey. The compounds used in this study were SEQ ID NOs 1013, 18, 19, 20 & 21, prepared in sterile saline (0.9%) at an initialconcentration of 0.625 and 2.5 mg/ml).

Male monkeys of at least 24 months old were used, and given free accessto tap water and 180 g of MWM(E) SQC SHORT expanded diet (Dietex France,SDS, Saint Gratien, France) was distributed daily per animal. The totalquantity of food distributed in each cage will be calculated accordingto the number of animals in the cage on that day. In addition, fruit orvegetables was given daily to each animal. The animals were acclimatedto the study conditions for a period of at least 14 days before thebeginning of the treatment period. During this period, pre-treatmentinvestigations were performed. The animals were dosed i.v. at a singledose of 0.25, 1.0 or 2.5 mg/kg (SEQ ID NO 10, 13, 18, and 21) or at asingle dose of 1.0 or 2.5 mg/kg (SEQ ID NO 19 and 20). The dose volumewas 0.4 mL/kg. 2 animals were used per group.

The dose formulations were administered once on Day 1. Animals wereobserved for a period of 7 weeks following treatment, and were releasedfrom the study on Day 51. Day 1 corresponds to the first day of thetreatment period. Clinical observations and body weight and food intake(per group) will be recorded prior to and during the study.

Blood was sampled and analyses performed at the following time points:

Study Day Parameters −8 RCP, L, Apo-B, PCSK9*, OA −1 L, Apo-B, PCSK9*,PK, OA 1 Dosing 4 LSB, L, Apo-B, PCSK9*, OA 8 LSB, L, Apo-B, PCSK9*, PK,OA 15 RCP, L, Apo-B, PCSK9* PK, OA 22 LSB, L, Apo-B, PCSK9* PK, OA 29 L,Apo-B, PCSK9* PK, OA 36 LSB, L, Apo-B, PCSK9* PK, OA 43 L, PK, Apo-B,PCSK9* PK, OA 50 RCP, L, Apo-B, PCSK9* PK, OA RCP 0 routine clinicalpathology, LSB = liver safety biochemistry, PK = pharmacokinetics, OA =other analysis, L = Lipids.

The following parameters were determined for all surviving animals atthe occasions indicated below:

-   -   full biochemistry panel (complete list below)—on Days −8, 15 and        50,    -   liver Safety (ASAT, ALP, ALAT, TBIL and GGT only)—on Days 4, 8,        22 and 36,    -   lipid profile (Total cholesterol, HDL-C, LDL-C and        Triglycerides) and Apo-B only—on Days −1, 4, 8, 22, 29, 36, and        43.

Blood (approximately 1.0 mL) was taken into lithium heparin tubes (usingthe ADVIA 1650 blood biochemistry analyzer): Apo-B, sodium, potassium,chloride, calcium, inorganic phosphorus, glucose, HDL-C, LDL-C, urea,creatinine, total bilirubin (TBIL), total cholesterol, triglycerides,alkaline phosphatase (ALP), alanine aminotransferase (ALAT), aspartateaminotransferase (ASAT), creatine kinase, gamma-glutamyl transferase(GGT), lactate dehydrogenase, total protein, albumin, albumin/globulinratio.

Analysis of PCSK9 in blood: Blood samples for PCSK9 analysis werecollected from on Days −8, −1, 4, 8, 15, 22, 29, 36, 43 and 50. Venousblood (approximately 2 mL) was collected from an appropriate vein ineach animal into a Serum Separating Tube (SST) and allowed to clot forat least 60±30 minutes at room temperature. Blood was centrifuged at1000 g for 10 minutes under refrigerated conditions (set to maintain+4°C.). The serum will be transferred into 3 individual tubes and stored at−80° C. until analyzed at CitoxLAB France using an ELISA method(Circulex Human PCSK9 ELISA kit, CY-8079, validated for samples fromcynomolgus monkey).

Other Analysis: WO2011009697 provides the methods for the followinganalysis: qPCR, PCSK9 mRNA analysis. Other analysis includes PCSK9protein ELISA, serum Lp(a) analysis with ELISA (Mercodia No.10-1106-01), tissue and plasma oligonucleotide analysis (drug content),Extraction of samples, standard—and QC-samples, Oligonucleotide contentdetermination by ELISA.

The data for the PCSK9 targeting compounds is shown in the followingtable:

Values PCSK9 PCSK9 Max PCSK9 Max LDL-C for 2.5 protein protein effect(data effect (data mg/kg dose day 4 day 29 represent represent Compound(percent of (percent of percent of percent of SEQ ID pre-dose) pre-dose)pre-dose) pre-dose) 10 86 71.5 69% (d 15) 87% (d 29) 13 81 71 71% (d 29)84% (d 22) 18 57 42 42% (d 29) 71% (d 15) 21 80.5 56 55% (d 29) 84% (d15) 20 51 53 48% (d 4)  94% (D 8)  19 55 60 55% (d 4)  89% (D 4) 

There was no indication of hepatotoxicity or nephrotoxicity with thePCSK9 targeting compounds. Notably, the PCSK9-GalNAc compounds gave arapid and highly effective down regulation of PCSK9 which was maintainedover an extensive time period (entire length of the study), illustratingthat the GalNAc conjugated compounds are more effective, both in termsof a rapid initial knock-down, and long duration, indicating that theymay be dosed comparatively infrequently and at a lower dosage, ascompared to both the unconjugated parent compounds, and compounds usingalternative conjugation technology, such as cholesterol conjugation. SEQID NO 18 gave rapid and consistent down regulation of PCSK9 and LDL-Cthroughout the duration of the study (seen at day 34 at 2.5 mg/kg dose,with notable PCSK9 down-regulation seen 48 days after the administrationof the single 2.5 mg/kg dose where plasma PCSK9 protein level was 71% ofpre-dose).

Example 5: Liver and Kidney Toxicity Assessment in Rat

Compounds of the invention can be evaluated for their toxicity profilein rodents, such as in mice or rats. The following protocol may be used:Wistar Han Crl:WI(Han) were used at an age of approximately 8 weeks old.At this age, the males weighed approximately 250 g. All animals had freeaccess to SSNIFF R/M-H pelleted maintenance diet (SSNIFF SpezialdiatenGmbH, Soest, Germany) and to tap water (filtered with a 0.22 μm filter)contained in bottles. The dose level of 10 and 40 mg/kg/dose was used(sub-cutaneous administration) and dosed on days 1 and 8. The animalswere euthanized on Day 15. Urine and blood samples were collected on day7 and 14. A clinical pathology assessment was made on day 14. Bodyweight is determined prior to the study, on the first day ofadministration, and 1 week prior to necropsy. Food consumption per groupwas assessed daily. Blood samples were taken via the tail vein after 6hours of fasting. The following blood serum analysis ws performed:erythrocyte count, mean cell volume packed cell volume, hemoglobin, meancell hemoglobin concentration, thrombocyte count, leucocyte count,differential white cell count with cell morphology, reticulocyte count,sodium, potassium, chloride, calcium, inorganic phosphorus, glucose,urea, creatinine, total bilirubin, total cholesterol, triglycerides,alkaline phosphatase, alanine aminotransferase, aspartateaminotransferase, total protein, albumin, albumin/globulin ratio.Urinalysis was performed: α-GST, β-2 Microglobulin, Calbindin,Clusterin, Cystatin C, KIM-1, Osteopontin, TIMP-1, VEGF, and NGAL. Sevenanalytes (Calbindin, Clusterin, GST-α, KIM-1, Osteopontin, TIMP-1, VEGF)were quantified under Panel 1 (MILLIPLEX® MAP Rat Kidney ToxicityMagnetic Bead Panel 1, RKTX1MAG-37K). Three analytes (β-2 Microglobulin,Cystatin C, Lipocalin-2/NGAL) were quantified under Panel 2 (MILLIPLEX®MAP Rat Kidney Toxicity Magnetic Bead Panel 2, RKTX2MAG-37K). The assayfor the determination of these biomarkers' concentration in rat urineswas based on the Luminex xMAP® technology. Microspheres coated withanti-α-GST/β-2 microglobulin/calbindin/clusterin/cystacinC/KIM-1/osteopontin/TIMP-1/VEGF/NGAL antibodies were color-coded withtwo different fluorescent dyes. The following parameters wre determined(Urine using the ADVIA 1650): Urine protein, urine creatinine.Quantitative parameters: volume, pH (using 10-Multistix SG teststrips/Clinitek 500 urine analyzer), specific gravity (using arefractometer). Semi-quantitative parameters (using 10-Multistix SG teststrips/Clinitek 500 urine analyzer): proteins, glucose, ketones,bilirubin, nitrites, blood, urobilinogen, cytology of sediment (bymicroscopic examination). Qualitative parameters: Appearance, color.After sacrifice, the body weight and kidney, liver and spleen weight aredetermined and organ to body weight ratio calculated. Kidney and liversamples was taken and either frozen or stored in formalin. Microscopicanalysis was performed. The data for Kim-1 expression are shown in FIG.15, where it is demonstrated that all molecules except SEQ ID NO 4 had alower urinary kim-1 signal than SEQ ID NO 1, demonstrating improvedkidney safety vs. the original and previously characterized unconjugatedmolecule.

Example 6 Analysis of Cleavable Linkers

FAM-labelled antisense oligomers (ASOs) with different DNA/PO-linkerswere subjected to in vitro cleavage either in S1 nuclease extract (tablebelow), Liver or kidney homogenates or Serum.

# Seq (5′-3′) Cleavable linker (B) Conjugate (C) 35 GCattggtatTCA3PO-DNA (5′tca3′) FAM 36 GCattggtatTCA 2PO-DNA (5′ca3′) FAM 37GCattggtatTCA 1PO-DNA (5′a3′) FAM 38 GCattggtatTCA 3PO-DNA (5′gac3′) FAM39 GCattggtatTCA no FAM

Capital letters are LNA nucleosides (such as beta-D-oxy LNA), lower caseletters are DNA nucleosides. Subscript s represents a phosphorothioateinternucleoside linkages. LNA cytosines are optionally 5-methylcytosine. The FAM conjugate moiety is shown in FIG. 6 and the moleculesare shown in FIG. 7.

FAM-labelled ASOs 100 μM with different DNA/PO-linkers were subjected toin vitro cleavage by S1 nuclease in nuclease buffer (60 U pr. 100 μL)for 20 and 120 minutes (A). The enzymatic activity was stopped by addingEDTA to the buffer solution. The solutions were then subjected to AIEHPLC analyses on a Dionex Ultimate 3000 using an Dionex DNApac p-100column and a gradient ranging from 10 mM-1 M sodium perchlorate at pH7.5. The content of cleaved and non-cleaved oligonucleotide weredetermined against a standard using both a fluorosensce detector at 615nm and a uv detector at 260 nm.

Linker % cleaved after % cleaved after SEQ ID NO sequence 20 min S1 120min S1 39 — 2 5 37 a 29.1 100 36 ca 40.8 100 35 tca 74.2 100 38 gac 22.9n.d

Conclusion:

The PO linkers (or region B as referred to herein) results in cleavageof the conjugate (or group C), and both the length and/or the sequencecomposition of the linker can be used to modulate susceptibility tonucleolytic cleavage of region B. The Sequence of DNA/PO-linkers canmodulate the cleavage rate as seen after 20 min in Nuclease S1 extractSequence selection for region B (e.g. for the DNA/PO-linker) cantherefore also be used to modulate the level of cleavage in serum and incells of target tissues.

Liver and kidney homogenates and Serum were spiked with compound SEQ IDNO 35 to concentrations of 200 μg/g tissue. Liver and kidney samplescollected from NMRI mice were homogenized in a homogenisation buffer(0.5% Igepal CA-630, 25 mM Tris pH 8.0, 100 mM NaCl, pH 8.0 (adjustedwith 1 N NaOH). The homogenates were incubated for 24 hours at 37° C.and thereafter the homogenates were extracted with phenol-chloroform.The content of cleaved and non cleaved oligo in the extract from liverand kidney and from the serum were determined against a standard usingthe above HPLC method:

% cleaved after % cleaved after % cleaved after Linker 24 hrs liver 24hrs kidney 24 hours in Seq ID Sequence homogenate homogenate serum 35tca 83 95 0

Conclusion:

The PO linkers (or region B as referred to herein) results in theconjugate (or group C) being cleaved off, in liver or kidney homogenate,but not in serum. The susceptibility to cleavage in the assays shown inExample 6 may be used to determine whether a linker is biocleavable orphysiologically labile. Note that cleavage in the above assays refers tothe cleavage of the cleavable linker, the oligomer or region A shouldremain functionally intact.

Example 7: Knock Down of PCSK9 mRNA with Cholesterol Conjugates In Vivo

PCSK9 - Mouse specific compounds # Seq (5′-3′)(A) Cleavable Linker (B)Conjugate (C) 40 GTctgtggaaGCG no no 41 GTctgtggaaGCG no Cholesterol 42GTctgtggaaGCG 2PO-DNA (5′ca3′) Cholesterol 43 GTctgtggaaGCG2PO-DNA (5′ct3′) Cholesterol

NMRI mice were injected with a single dose saline or 10 mg/kgunconjugated LNA-antisense oligonucleotide (SEQ ID 40) or equimolaramounts of LNA antisense oligonucleotides conjugated to Cholesterol withdifferent linkers and sacrificed at days 1-10 according to Tab. 5.

RNA was isolated from liver and kidney and subjected to qPCR with PCSK9specific primers and probe to analyze for PCSK9 mRNA knockdown. Theresults are shown in FIG. 14.

Conclusions:

Cholesterol conjugated to an PCSK9 LNA antisense oligonucleotide with alinker composed of 2 DNA with Phophodiester-backbone (SEQ ID NO 42 andSEQ ID NO 43) showed an enhanced liver knock down of PCSK9 (FIG. 14)compared to the unconjugated compound (SEQ ID NO 40), as well ascompared to Cholesterol conjugates with stable linker (SEQ ID NO 41).

Materials and Methods:

Experimental Design:

Compound Body Group Animal id No. of Animal strain/ Dose level per Conc.at dose weight Sacrifice Part no. no. Animals gender/feed day vol. 10ml/kg Adm. Route Dosing day day day A 1 1-3 3 NMRI/♀/Chow Saline — iv 00, 1 1 2 4-6 3 NMRI/♀/Chow SEQ ID NO 40   1 mg/ml iv 0 0, 1 1 10 mg/kg 37-9 3 NMRI/♀/Chow SEQ ID NO 41 1.13 mg/ml iv 0 0, 1 1 equimolar 11.3mg/kg 5 13-15 3 NMRI/♀/Chow SEQ ID NO 42 1.27 mg/ml iv 0 0, 1 1equimolar 12.7 mg/kg 6 16-18 3 NMRI/♀/Chow SEQ ID NO 43 1.27 mg/ml iv 00, 1 1 equimolar 12.7 mg/kg B 7 19-21 3 NMRI/♀/Chow Saline — iv 0 0, 3 38 22-24 3 NMRI/♀/Chow SEQ ID NO 40   1 mg/ml iv 0 0, 3 3 10 mg/kg 925-27 3 NMRI/♀/Chow SEQ ID NO 41 1.13 mg/ml iv 0 0, 3 3 equimolar 11.3mg/kg 11 31-33 3 NMRI/♀/Chow SEQ ID NO 42 1.27 mg/ml iv 0 0, 3 3equimolar 12.7 mg/kg 12 34-36 3 NMRI/♀/Chow SEQ ID NO 43 1.27 mg/ml iv 00, 3 3 equimolar 12.7 mg/kg C 13 37-39 3 NMRI/♀/Chow Saline — iv 0 0, 77 14 40-42 3 NMRI/♀/Chow SEQ ID NO 40   1 mg/ml iv 0 0, 7 7 10 mg/kg 1543-45 3 NMRI/♀/Chow SEQ ID NO 41 1.13 mg/ml iv 0 0, 7 7 equimolar 11.3mg/kg 17 49-51 3 NMRI/♀/Chow SEQ ID NO 42 1.27 mg/ml iv 0 0, 7 7equimolar 12.7 mg/kg 18 52-54 3 NMRI/♀/Chow SEQ ID NO 43 1.27 mg/ml iv 00, 7 7 equimolar 12.7 mg/kg D 19 55-57 3 NMRI/♀/Chow Saline — iv 0 0, 7,10 10 20 58-60 3 NMRI/♀/Chow SEQ ID NO 40   1 mg/ml iv 0 0, 7, 10 10 10mg/kg 21 61-63 3 NMRI/♀/Chow SEQ ID NO 41 1.13 mg/ml iv 0 0, 7, 10 10equimolar 11.3 mg/kg 24 70-72 3 NMRI/♀/Chow SEQ ID NO 42 1.27 mg/ml iv 00, 7, 10 10 equimolar 12.7 mg/kg A 25 73-75 3 NMRI/♀/Chow Saline — iv 00, 1 1

Dose administration. NMRI female animals, app. 20 g at arrival, weredosed with 10 ml per kg BW (according to day 0 bodyweight) i.v. of thecompound formulated in saline or saline alone according to according tothe table above.

Sampling of Liver and Kidney Tissue.

The animals were anaesthetized with 70% CO₂-30% O₂ and sacrificed bycervical dislocation according to Table 4. One half of the large liverlobe and one kidney were minced and submerged in RNA later.

Total RNA was extracted from maximum 10 mg of tissue homogenized bybead-milling in the presence of MagNA Pure LC RNA Isolation Tissuebuffer (Roche cat. no 03 604 721 001) using the MagNa Pure 96 CellularRNA Large Volume Kit (Roche cat no. 5467535001), according to themanufacturer's instructions. First strand synthesis was performed usingReverse Transcriptase reagents from Ambion according to themanufacturer's instructions.

For each sample 0.5 μg total RNA was adjusted to (10.8 μl) with RNasefree H₂O and mixed with 2 μl random decamers (50 μM) and 4 μl dNTP mix(2.5 mM each dNTP) and heated to 70° C. for 3 min after which thesamples were rapidly cooled on ice. 2 μl 10× Buffer RT, 1 μl MMLVReverse Transcriptase (100 U/μl) and 0.25 μl RNase inhibitor (10 U/μl)were added to each sample, followed by incubation at 42° C. for 60 min,heat inactivation of the enzyme at 95° C. for 10 min and then the samplewas cooled to 4° C. cDNA samples were diluted 1:5 and subjected toRT-QPCR using Taqman Fast Universal PCR Master Mix 2× (AppliedBiosystems Cat #4364103) and Taqman gene expression assay (mPCSK9,Mn00463738_m1 and mActin #4352341E) following the manufacturers protocoland processed in an Applied Biosystems RT-qPCR instrument (7500/7900 orViiA7) in fast mode.

Example 8: Non-Human Primate Study; Multiple Injections s.c

The objective of this non-human primate study was to assess efficacy andsafety of the anti-PCSK9 compounds in a repeat administration setting,when compounds were administered by subcutaneous injection (s.c.). Thecompounds used in this study were SEQ ID NOs 2, 3, 18, and 19, preparedin sterile saline (0.9%) at an initial concentration of 0.625 and 2.5mg/ml.

Female cynomolgus monkeys of at least 24 months old were used, and givenfree access to tap water and 180 g of OWM(E) SQC SHORT expanded diet(Dietex France, SDS, Saint Gratien, France) was distributed daily peranimal. In addition, fruit or vegetables were given daily to eachanimal. The animals were acclimated to the study conditions for a periodof at least 14 days before the beginning of the treatment period. Duringthis period, pre-treatment investigations were performed. The animalswere dosed s.c. once per week for four weeks at a dose of 0.5 mg/kg (SEQID NO 2, 3, 18, and 19) or 1.5 mg/kg/injection (SEQ ID NO 18 and 19),with four injections total over a period of four weeks. The dose volumewas 0.4 mL/kg/injection. Six animals were used per group. After thefourth and final dose animals were observed for a week after which halfthe animals were sacrificed in order to study liver apoB transcriptregulation, lipid parameters, liver and kidney histology, and liver andkidney tissue distribution. Day 1 corresponds to the first day of thetreatment period. Clinical observations and body weight and food intake(per group) was recorded prior to and during the study.

Blood and tissues were sampled and analysed at the following timepoints:

Study Day Parameters −10  L, Apo-B, OA −5 LSB, L, Apo-B, OA −1 RCP, L,Apo-B, PK, OA  1 Dosing  8 LSB, L, Apo-B, PK, OA pre-dose  8 Dosing 15LSB, L, Apo-B, PK, OA pre-dose 15 Dosing 22 LSB, L, Apo-B, PK, OApre-dose 22 Dosing 29 RCP, PK, OA + necropsy main 36 LSB, L, Apo-B, PK,OA (recovery animals) 43 RCP, PK, Apo-B, PK, OA (recovery animals) 50LSB, L, Apo-B, PK, OA (recovery animals) 57 LSB, L, Apo-B, PK, OA(recovery animals) 64 LSB, L, Apo-B, PK, OA (recovery animals) 71 LSB,L, Apo-B, PK, OA (recovery animals) 78 RCP, L, Apo-B, PK, OA + necropsyrecovery (recovery animals)

RCP: routine clinical pathology, LSB: liver safety biochemistry, PK:pharmacokinetics, OA: other analyses, L: lipids

Blood (approximately 1.0 mL) was taken into lithium heparin tubes (usingthe ADVIA 1650 blood biochemistry analyser) analyzing sodium, potassium,chloride, calcium, inorganic phosphorus, glucose, HDL-C, LDL-C, urea,creatinine, total bilirubin (TBIL), total cholesterol, triglycerides,alkaline phosphatase (ALP), alanine aminotransferase (ALAT), aspartateaminotransferase (ASAT), creatine kinase, gamma-glutamyl transferase(GGT), lactate dehydrogenase, total protein, albumin, albumin/globulinratio.

Analysis of blood: Blood samples for ApoB analysis was collected fromGroup 1-16 animals only (i.e. animals treated with anti-ApoB compounds)on Days −8, −1, 4, 8, 15, 22, 29, 36, 43 and 50. Venous blood(approximately 2 mL) was collected from an appropriate vein in eachanimal into a Serum Separating Tube (SST) and allowed to clot for atleast 60±30 minutes at room temperature. Blood was centrifuged at 1000 gfor 10 minutes under refrigerated conditions (set to maintain+4° C.).The serum was transferred into 3 individual tubes and stored at −80° C.until analysis of ApoB protein by ELISA.

Other Analysis: WO2010142805 provides the methods for the followinganalysis: qPCR, ApoB mRNA analysis. Other analysis includes, serum Lp(a)analysis with ELISA (Mercodia No. 10-1106-01), tissue and serumoligonucleotide analysis (drug content), Extraction of samples,standard—and QC-samples, Oligonucleotide content determination by ELISA.

The intended pharmacology for an anti-PCSK9 oligonucleotide is reductionin LDL cholesterol by a reduction of PCSK9 protein in circulation(“serum PCSK9”). The GalNAc conjugated molecules demonstrated enhancedefficacy compared to unconjugated molecules when studying both serumPCSK9 and LDL cholesterol (FIG. 16 and FIG. 17). FIG. 16 illustratesthat four weekly injections of 0.5 mg/kg/injection of the unconjugatedSEQ ID NO 2 had only minor effects on serum PCSK9 and LDL cholesterol,whereas the GalNAc conjugate of the same LNA gap-mer (SEQ ID 18) had apotent reducing effect on both serum PCSK9 and LDL cholesterol. The samerelation was noted when comparing data for multiple injections of SEQ IDNO 3 and SEQ ID NO 19 (FIG. 17): only minor effects of the unconjugatedmolecule and potent down-regulation of serum PCSK9 and LDL cholesterolby the corresponding GalNAc conjugate (SEQ ID NO 19). It should be notedthat effects of SEQ ID 18 and 19 on serum PCSK9 and LDL cholesterol weredose dependent and with long duration of action, with serum PCSK9 andLDL cholesterol lower than average baseline levels for at least sevenweeks after the last injection (last injection day 22, data illustratedfor the recovery period up to day 71).

Liver and kidney oligonucleotide content was analysed one week afterlast injection, i.e. day 29 of the study. Oligonucleotide content wasanalysed using hybridization ELISA (essentially as described in Lindholmet al, Mol Ther. 2012 February; 20(2):376-81), using SEQ ID NO 2 toprepare a standard curve for samples from animals treated with SEQ ID NO2 and SEQ ID NO 18, after having controlled that there was no change inresult if the (conjugated) SEQ ID NO 18 was used for preparation ofstandard curve. In the same manner, SEQ ID NO 3 was used for preparationof standard curve for SEQ ID NO 3 and SEQ ID NO 19 after controllingthat there was no difference in result if SEQ ID NO 19 was used forpreparation of standard curve for ELISA analysis of those samples.

Oligonucleotide content in tissues one week after last injection LiverKidney (μg oligonucleotide/ (μg oligonucleotide/ Liver/ g wet tissue) gwet tissue) kidney Average SD Average SD ratio SEQ ID NO 2, 0.260 0.1430.3 4.8 0.008 4 × 0.5 mg/kg SEQ ID NO 18, 3.57 0.61 11.5 2.5 0.310 4 ×0.5 mg/kg SEQ ID NO 18, 18.8 1.7 26.8 6.6 0.701 4 × 1.5 mg/kg SEQ ID NO3, 0.149 0.059 38.2 0.72 0.004 4 × 0.5 mg/kg SEQ ID NO 19, 2.72 0.6916.3 1.5 0.167 4 × 0.5 mg/kg SEQ ID NO 19, 12.2 3.44 41.2 6.5 0.296 4 ×1.5 mg/kg

As illustrated in the table above, conjugation of SEQ ID NO 2 and SEQ IDNO 3 resulted in higher liver/kidney ratios for the conjugated molecules(SEQ ID NO 18 and SEQ ID 19) than for the corresponding unconjugatedmolecules one week after last injection when animals were injected s.c.once/week for four weeks. Given that signs of tubulotoxicity has beendemonstrated with other unconjugated anti-PCSK9 molecules (such as SEQID NO 1, as illustrated in FIG. 15), and given that liver is the targetorgan for anti-PCSK9 treatment, a shift to a higher liver/kidney ratiois expected to result in increased safety with the conjugated SEQ ID NO18 and 19 compared to the unconjugated SEQ ID NO 2 and 3.

As illustrated in FIG. 16 and FIG. 17, SEQ ID NO 18 and 19 were dosed atpharmacology relevant levels. Clinical chemistry profiles of the sameanimals during the treatment period and the recovery phase demonstratedno clinically relevant increases in liver or kidney safety parameters.

The invention claimed is:
 1. A method of manufacturing a compoundconsisting of an antisense oligomer that is 16 contiguous nucleotides inlength, the method comprising chemically synthesizing the compound usingsequential solid phase oligonucleotide synthesis, wherein the sequenceof the antisense oligomer is 100% complementary to the sequence of SEQID NO: 31, wherein the antisense oligomer is a gapmer comprising atleast one LNA unit, and wherein the antisense oligomer targets mRNAencoding PCSK9.
 2. The method of claim 1, wherein the antisense oligomeris an intermediate.
 3. The method of claim 1, wherein the sequence ofthe antisense oligomer is SEQ ID NO:
 26. 4. The method of claim 1,wherein the LNA is oxy-LNA, thio-LNA, amino-5 LNA, 5′-methyl-LNA, ENA,cET, cMOE or a combination thereof.
 5. The method of claim 1, whereinthe LNA is a stereoisomer in the beta-D-configuration or the alpha-Lconfiguration.
 6. The method of claim 1, wherein the antisense oligomercomprises at least one cET unit.
 7. The method of claim 1, wherein theantisense oligomer comprises six or seven LNA units.
 8. The method ofclaim 7, wherein every LNA unit in the antisense oligomer is abeta-D-oxy LNA unit or every LNA unit in the antisense oligomer is analpha-L-oxy-LNA unit.
 9. The method of claim 7, wherein every LNA unitin the antisense oligomer is a stereoisomer in the same configuration.10. The method of claim 1, wherein the sequence of the antisenseoligomer comprises at least one phosphorothioate, phosphorodithioate, orboranophosphate internucleoside linkage.
 11. The method of claim 10,wherein all the internucleoside linkages are phosphorothioate.
 12. Themethod of claim 10, wherein one or more of the inernucleoside linkagescomprises a chiral center in the R conformation and/or in the Sconformation.
 13. The method of claim 1, wherein the sequence of theantisense oligomer is SEQ ID NO:
 2. 14. The method of claim 1, whereinthe sequence of the antisense oligomer is SEQ ID NO:
 3. 15. The methodof claim 1, wherein the compound and an oligomer of SEQ ID NO:31 canform a duplex with increased thermal stability with respect to acorresponding duplex comprising the corresponding antisense oligomerwithout LNA.
 16. A method of manufacturing an antisense oligonucleotideconjugate comprising covalently attaching at least one non-nucleotide ornon-polynucleotide moiety covalently to an antisense oligomer that is 16contiguous nucleotides in length directly or via a linker positionedbetween the antisense oligomer sequence and the non-nucleotide ornon-polynucleotide moiety, wherein the sequence of the antisenseoligomer is 100% complementary to the sequence of SEQ ID NO: 31, whereinthe antisense oligomer is a gapmer comprising at least one LNA unit, andwherein the antisense oligonucleotide conjugate targets mRNA encodingPCSK9.
 17. The method of claim 16, wherein the non-nucleotide ornon-polynucleotide moiety is a liver targeting moiety that is attachedto the 5′-end or to the 3′-end of the antisense oligomer.
 18. The methodof claim 17, wherein the liver targeting moiety is linked to theantisense oligomer via a linker.
 19. The method of claim 18, wherein thelinker is a biocleavable linker.
 20. The method of claim 17, wherein theliver targeting moiety comprises a carbohydrate conjugate moietycomprising a carbohydrate or a carbohydrate group.
 21. The method ofclaim 20, wherein the carbohydrate is selected from the group consistingof galactose, lactose, N-acetylgalactosamine (GalNAc), mannose,mannose-6-phosphate, and combinations thereof.
 22. The method of claim20, wherein the carbohydrate conjugate moiety is not a linearcarbohydrate polymer.
 23. The method of claim 20, wherein thecarbohydrate conjugate moiety is a carbohydrate group comprising 1 2, 3,or 4 carbohydrate moieties.
 24. The method of claim 23, wherein thecarbohydrate moieties are identical or non-identical.
 25. The method ofclaim 23, wherein the carbohydrate moieties are non-identical.
 26. Themethod of claim 20, wherein the carbohydrate conjugate moiety comprisesat least one asialoglycoprotein receptor targeting conjugate moiety. 27.The method of claim 26, wherein the asialoglycoprotein receptortargeting conjugate moiety comprises a GalNAc cluster.
 28. The method ofclaim 26, wherein the asialoglycoprotein receptor targeting conjugatemoiety comprises a monovalent, divalent, trivalent, or tetravalentGalNAc cluster.
 29. The method of claim 27, wherein the GalNAc clusteris a trivalent GalNAc cluster.
 30. The method of claim 29, wherein eachGalNAc in the trivalent GalNAc cluster is attached to a branch pointgroup via a spacer.
 31. The method of claim 30, wherein the branch pointgroup comprises di-lysine.
 32. The method of claim 30, wherein thespacer comprises a PEG spacer.
 33. The method of claim 18, wherein thelinker comprises a C6 to C12 amino alkyl group or a biocleavablephosphate nucleotide linker comprising between 1 to 6 nucleotides. 34.The method of claim 29, wherein the trivalent GalNAc cluster comprisesConj 2a:


35. The method of claim 16, wherein the non-nucleotide ornon-polynucleotide moiety is covalently attached to the antisenseoligomer via a covalent bond.
 36. The method of claim 16, wherein theantisense oligonucleotide conjugate is SEQ ID NO:
 18. 37. The method ofclaim 16, wherein the antisense oligonucleotide conjugate is SEQ ID NO:19.
 38. The method of claim 16, wherein covalently attaching the atleast one non-nucleotide or non-polynucleotide moiety to the antisenseoligomer comprises: (i) chemically synthesizing the antisense oligomer;and, (ii) adding by chemical synthesis or conjugation the at least onenon-nucleotide or non-polynucleotide moiety to the antisenseoligonucleotide conjugate.
 39. The method of claim 38, wherein adding bychemical synthesis or conjugation the at least one non-nucleotide ornon-polynucleotide moiety to the antisense oligonucleotide conjugatecomprises: (i) incorporating by chemical synthesis or conjugation atleast one non-nucleotide or non-polynucleotide moiety to the antisenseoligonucleotide conjugate; (ii) incorporating by chemical synthesis orconjugation at least one linker to the antisense oligonucleotideconjugate; (iii) incorporating by chemical synthesis or conjugation atleast one branching point to the antisense oligonucleotide conjugate;(iv) incorporating by chemical synthesis or conjugation at least onespacer to the antisense oligonucleotide conjugate; or, (v) a combinationthereof.
 40. The method of claim 39, wherein at least one linker isinterposed between the antisense oligomer and a branching point.
 41. Themethod of claim 39, wherein at least one branching point is interposedbetween a linker and at least one non-nucleotide or non-polynucleotidemoiety.
 42. The method of claim 39, wherein at least one, two, or threenon-nucleotide or non-polynucleotide moieties are attached to abranching point.
 43. The method of claim 39, wherein at least one PEGspacer is interposed between each of the one, two, or threenon-nucleotide or non-polynucleotide moieties and a branching point. 44.A method of manufacturing an antisense oligonucleotide conjugate of SEQID NO: 18

wherein a superscript ^(L) identifies a beta-D-oxy LNA unit, a ^(Me)Cidentifies a 5-methylcytosine unit, a subscript _(s) identifies aphosphorothioate internucleoside linkage, and wherein

is a Conj 2a asialoglycoprotein receptor targeting conjugate moiety

and wherein the antisense oligonucleotide conjugate targets mRNAencoding PCSK9, the method comprising covalently attaching the Conj 2aasialoglycoprotein receptor targeting conjugate moiety to the 5′ end ofan antisense oligomer of SEQ ID NO:
 2. 45. A method of manufacturing anantisense oligonucleotide conjugate of SEQ ID NO: 19

wherein a superscript ^(L) identifies a beta-D-oxy LNA unit, a ^(Me)Cidentifies a 5-methylcytosine unit, a subscript _(s) identifies aphosphorothioate internucleoside linkage, and wherein

is a Conj 2a asialoglycoprotein receptor targeting conjugate moiety

and wherein the antisense oligonucleotide conjugate targets mRNAencoding PCSK9, the method comprising covalently attaching the Conj 2aasialoglycoprotein receptor targeting conjugate moiety to the 5′ end ofan antisense oligomer of SEQ ID NO: 3.